Protection against Acetaminophen Hepatotoxicity by a Single Dose of Clofibrate: Effects on Selective Protein Arylation and Glutathione Depletion

Previous reports demonstrated that repeated administration ofperoxisome proliferators protects against acetaminophen (APAP)hepatotoxicity in mice. This protection was associated witha decrease in APAP's selective protein arylation and glutathionedepletion. This study was conducted to determine if a singledose of clofibrate (CFB), rather than repeated doses, wouldsimilarly prevent APAP toxicity. CD-1 male mice received a singledose of 500 mg CFB/kg and controls were given corn oil 24 hrprior to APAP challenge. After an 18-hr fast, mice were challengedwith 800 mg APAP/kg (in 50% propylene glycol) and killed at4 or 12 hr. Other mice similarly pretreated were killed withoutAPAP challenge. The results showed that pretreatment with asingle CFB dose significantly decreased APAP-induced hepatotoxicity.At 12 hr after APAP plasma sorbitol dehydrogenase activity andthe severity of hepatocellular necrosis were decreased in CFBpretreated mice. Surprisingly, no differences in hepatic nonproteinsulfhydryl (NPSH) depletion or selective arylation of targetproteins in cytosol were observed at 4 hr after APAP challenge.Neither did a single dose of CFB significantly alter hepaticNPSH content prior to APAP challenge. These results indicatethat protection against APAP hepatotoxicity by CFB does notrequire repeated administration, and the absence of significantalterations in APAP's selective protein arylation or glutathionedepletion suggests that the protection against APAP hepatotoxicityafter a single treatment with CFB may differ mechanisticallyfrom the protection observed after repeted CFB dosing.     

Evidence Suggesting the 58-kDa Acetaminophen Binding Protein Is a Preferential Target for Acetaminophen Electrophile

Acetaminophen is an analgesic and antipyretic which causes livertoxicity in humans and experimental animals with overdose. Acetaminophen(APAP) covalent binding to a cytosolic protein of approximately58 kDa (58–ABP) has been associated with target organtoxicity. Since hepatic content of 58–ABP varies, studieswere conducted to determine if this influences APAP bindingto other target proteins. In the liver, the amount of 58–ABPvaried with individual male CD-1 mice, but in kidneys of thesame mice there was no such variability in 58–ABP content.All male A/J mice tested had comparatively little detectable58–ABP in liver cytosol. Similarly, female CD–1mice had low 58–ABP content compared to males; however,administration of testosterone propionate to females significantlyincreased 58-ABP content in liver cytosol. At 4 hr after challengeof mice from the above-described groups with 600 mg APAP/kg,cytosolic covalent binding to proteins was determined by Westernblot analysis with anti–APAP antibody. The Western blotswere then stripped of antibody and blocking agents and reprobedwith antibody prepared against purified 58–ABP (anti–58–ABP).In the liver, the level of APAP bound to the 58-ABP target correspondedwith 58–ABP content. In cases where 58–ABP was poorlyexpressed, APAP adducts to other protein targets were more prominentlydetected. In the kidneys of the male CD–1 mice 58–ABParylation by APAP varied little among animals, reflecting therelatively consistent levels of renal 58–ABP. These datasuggest that binding to the 58–ABP may spare other potentialtargets of APAP electrophile attack and support a role of the58–ABP as a preferred target of APAP electrophile in cytosol.