Title

Characterization of the AP56/SP56-acetaminophen binding protein and its nuclear translocation

Date of Completion

January 2000

Keywords

Health Sciences, Pharmacology|Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

Acetaminophen (APAP) overdose can result in liver injury and even death. APAP-induced hepatotoxicity is associated with depletion of hepatic glutathione, (GSH) and covalent binding (CVB) of its metabolite to specific proteins. In liver cytosol, the predominant target for APAP CVB is a 56-kDa protein (AP56/SP56), and binding to this protein correlates with APAP hepatotoxicity. After exposure to a hepatotoxic APAP dose, AP56/SP56 is detected in liver nuclei in advance of any detectable nuclear perturbations. Thus, the main focus of this dissertation work was to further characterize AP56/SP56 nuclear translocation and to examine the relationship between translocation and GSH depletion, CVB, and exposures that ultimately result hepatotoxicity. To examine these relationships, murine liver nuclear fractions were resolved by SDS-PAGE and analyzed for AP56/SP56 content by western analysis with AP56/SP56 antisera. AP56/SP56 translocation was associated with APAP doses that resulted in hepatocellular injury, but was not associated with a single non-hepatotoxic treatment. This suggested that detection of nuclear AP56/SP56 after APAP may be a response that is common to treatments that result in hepatotoxicity. Therefore, translocation was examined after exposure to several other toxicants. AP56/SP56 was not detected in liver nuclear fractions from mice exposed to chloroform or carbon tetrachloride, but was detected after 3,5-dimethyl acetaminophen exposure. Thus, translocation may not be common to all hepatotoxicants. Next, the role of CVB and translocation was examined. Repeated sub-toxic APAP doses produced an equivalent amount of CVB to AP56/SP56 and other cytosolic proteins as observed after a single toxic APAP dose. Translocation was not detected after the repeat dose regimen, but was detected after the single toxic dose. To determine if translocation is mediated by GSH depletion, nuclear fractions were isolated from mice administered GSH depleting agents—AP56/SP56 was not detected after treatment with these agents. These data demonstrate that AP56/SP56 is not mediated solely by CVB or GSH depletion and is not common to all hepatotoxicants. ^ To further study 56-SBP translocation, a recombinant AP56 was engineered and expressed in E. coli that cross reacts with AP56/SP56 antisera. Furthermore, purified protein has been obtained used nickel column chromatography. ^