Many hepatotoxicants like acetaminophen, chloroform, carbon tetrachloride, halothane, and thioacetamide cause hepatotoxicity through covalent binding of their reactive metabolites to proteins. The covalent binding to proteins may lead to dysfunction of critical proteins such as enzymes, transporters, receptors, and regulatory molecules. Because most reactive metabolites covalently bind to tissue macromolecules and tend to be unstable, they can not be isolated, and direct quantitation of the formation of reactive metabolites is not possible. Measuring their covalent binding to proteins offers a convenient way to estimate the amount of reactive metabolite formation. Such estimates have been used to quantify the bioactivation-based injury due to such hepatotoxicants. There are various methods by which covalent binding may be measured. This unit describes a protocol in which a radiolabeled compound can be utilized to measure covalent binding. Alternate protocols involve immunoblotting and immunohistochemistry. The time and method of measuring covalent binding play an important role in the evaluation.
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