Reactive metabolites glutathione adducts

In summary, a growing body of evidence suggests that at least part of the chronic toxicity of buta-1,3-diene is caused by reactive metabolites that bear an epoxy or ,/Tunsaluralcd carbonyl group. These metabolites form adducts with protective (glutathione) or critical (e.g., nucleic acids) endogenous nucleophiles [163] [166] [167],... 

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]. 

FIGURE 37.2. Metabolism and mechanism of acetaminophen toxicity. Bioactivation of acetaminophen by P450 enzymes results in the formation of the reactive intermediate (NAPQI) which forms covalent protein adducts with glutathione which is then converted to mercapturic acid. When the amount of the reactive metabolite formed exceeds the glutathione available for binding, the excess metabolite binds to tissue molecules resulting in centrilobular hepatic necrosis. 

Chen, L. J., Hecht, S. S., Peterson, L. A. Characterization of amino acid and glutathione adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan. Chem. Res. Toxicol. 1997, 10, 866-874. 

In vitro systems combined with LC-MS/MS can also be used for the prediction of problematic or reactive metabolites. As shown in Figure 12.3, Bertrand et al. [16] have used an LC-MS/MS assay to look for glutathione (GSH) adducts of a test compound. As they point out this technique can be successful without the need for a radio-labeled compound and is therefore applicable in the early drug-discovery process. 

No indication for a reactive metabolite pathway. No glutathione adducts identified. No indication of protein adducts in human hepatocytes or subcellular fractions at concentrations up to 300 pM... 

Figure 10.3 Formation and possible fates of a reactive metabolite. Once formed at the active site of a CYP, it might dissociate and go on to form a covalent adduct with glutathione (GSH) or nucleophilic groups in cellular components. Alternately, reaction at the active site of the CYP can lead to quasi-irreversible inhibition by complexation with the iron atom of heme or to irreversible CYP inhibition by covalent bond formation either with the heme unit or with a nucleophilic component of the CYP protein.

Reactive Metabolites or Intermediates Reactive metabolites are chemically unstable and often rapidly bind to proteins or glutathione (GSH). In general, GSH adducts are not present at significant levels in circulation, because they undergo rapid elimination via direct biliary excretion or further metabolism to mercapturic acids that are excreted into urine. Reactive metabolites can be monitored via quantitative analysis of their GSH adducts or mercapturic acids in excreta. However, it is not practical to test the toxicity of reactive metabolites on animals directly since the synthesis and dosing preparation of reactive metabolites are extremely difficult or impossible due to their chemical instability (Davis-Bruno and Atrakchi, 2006). 

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