High-Mobility Group Box-1 Protein and Keratin-18, Circulating Serum Proteins Informative of Acetaminophen-Induced Necrosis and Apoptosis In Vivo

Abstract

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.

Redox Modification of Cysteine Residues Regulates the Cytokine Activity of High Mobility Group Box-1 (HMGB1)

“…HMGB1 was isolated by immunoprecipitation from mice sera as described previously (12). HMGB1 preparations then were separated by nonreducing SDS PAGE and protein bands corresponding to the molecular weight of HMGB1 were excised.…”

Intraperitoneal administration of high doses of polyethylene glycol (PEG) causes hepatic subcapsular necrosis and low-grade peritonitis with a rise in hepatic biomarkers

“…Alanine transaminase activity (ALT) was determined in all animals using ThermoTrace Infinity ALT Liquid stable reagent according to the manufacturer's instructions. In 12 male CD-1 mice euthanased at 5 and 8 h after PEG administration, miRNA levels were measured using a Taqman-based quantitative polymerase chain reaction (PCR), as previously described (Antoine et al, 2009). …”

“…From all animals, multiple sections from the left and median hepatic lobes were prepared from formalin- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Immunohistology for the demonstration of TNF-α, IL-6, cleaved caspase 3 and proliferating cell nuclear antigen (PCNA) was performed on selected CD-1 mice euthanased at 3, 5, 8 and/or 24 h after dosing, following previously described protocols (Antoine et al, 2009). …”

Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives

“…This led to the recent development of a point-of-care test for the accelerated diagnosis of APAP overdose in patients [243]. Other biomarkers such as miR-122 [244–247], full-length cytokeratin-18 [247–250], high mobility group box 1 (HMGB1) protein [247–249, 251], and argininosuccinate synthetase (ASS) [252] are generally considered indicators of mainly necrotic cell death [253]. On the other hand, increased plasma levels of the caspase-cleaved form of cytokeratin-18 [247, 248, 250, 254, 255] and activated caspases detected as enzyme activity and/or the protein by western blotting [164, 247, 249, 254], are markers of apoptotic cell death.…”