Proteomic analysis of circulating EVs isolated from APAP-exposed mice and confirmation of various proteins by immunoblot analysis.

<p>(A) Proteomic analysis of EV proteins isolated from Control or APAP-exposed mice using 2D LC-MS/MS. Differentially expressed proteins were identified by bioinformatic analysis, as described. (B) Classification based on the subcellular location of the identified proteins. Comparison of proteomic data from the EVs in this study with those of rat primary hepatocytes, as reported previously by our group[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172463#pone.0172463.ref030" target="_blank">30</a>]. (C) The 380 proteins in EVs from control plasma and 431 proteins in EVs from APAP plasma were classified by tissuespecific origin and categorized according to the DAVID program. Some proteins are classified to more than two organs. (D) Comparison of differentially expressed proteins in circulating EVs from APAP-treated groups with the previously reported results[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172463#pone.0172463.ref039" target="_blank">39</a>] from hepatocytes treated with APAP, based on biological process analysis. (E) Confirmation of liver-specific proteins, inflammation-related proteins, and exosomal marker proteins in circulating EVs by immunoblot analysis, as indicated. (F) Protein levels of ASS1 and CES1 were measured in whole liver lysates from the indicated groups.</p

Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

<div><p>Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with <i>N</i>-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.</p></div

Analysis of liver- or muscle-specific proteins in circulating EVs from mice with liver or muscle injury.

<p>Wild-type male BALB/c mice (6 weeks old) were treated with a single ip injection of APAP (300 mg/kg) or intramuscular injection with 0% or 0.5% BPVC into the right and left tibialis anterior muscles and were sacrificed at 24 h post treatment (n = 10/group). (A) Representative H&E staining of formalin-fixed muscle sections for all indicated groups. (B) Plasma ALT and AST levels. (C) Analysis of the total protein amounts in circulating EVs isolated from the indicated groups. (D) Immunoblot analyses of liver-specific or muscle-specific proteins in circulating EVs from each group, as indicated. (E) The amounts of ALB or STN1 in circulating EVs from each group were respectively measured by ELISA specific for mouse proteins. *<i>P</i> < 0.05.</p

The number and protein content in purified EVs from cultured cells were increased by APAP.

<p>Rat primary hepatocytes were exposed to APAP (20 mM APAP) for 24 h (n = 4/group). (A) A representative electron microscopic image of EVs isolated from culture media after primary hepatocytes were exposed to saline (CON) or APAP. (B) Analysis of the size distribution of CON- and APAP-derived EVs. (C) Representative immunoblot analysis of exosome marker proteins in EVs isolated from culture media. (D) Rat primary hepatocytes were exposed to saline (CON), APAP at the IC₃₀ (10 mM APAP), or APAP at the IC₅₀ (20 mM APAP) for 24 h, and the EVs isolated from the conditioned media (n = 10/group). The numbers and protein amounts in isolated EVs were determined by NanoSight analysis and protein quantification, respectively. (E and F) HepG2 and Hep3B cells were treated with APAP (at the IC₃₀ or IC₅₀) for 24 h. EVs were isolated from the conditioned media and analyzed using NanoSight analysis and protein quantification, respectively. *<i>P</i> < 0.05.</p

Analysis of total proteins and albumin amounts in circulating EVs after prevention of liver injury by NAC or GSH.

<p>Acute liver injury was induced in BALB/c male mice by a single i.p. injection of APAP (300 mg/kg), and some of the mice received 10 mL/kg saline, 100 mg/kg NAC, or 200 mg/kg GSH intravenously 1.5 h after APAP administration (n = 10/group). (A) Representative H&E staining of formalin-fixed liver sections. (B) Plasma ALT levels. (C) Protein amounts in circulating EVs derived from each indicated group were calculated. (D) Detection of liver-specific proteins in circulating EVs by immunoblot analyses, as indicated. (E) The amounts of mouse ALB in EVs from each group were measured by ELISA. (F) Hep3B and primary hepatocyte cells were exposed to APAP (IC₅₀) without or with NAC (5 mM) for 24 h (n = 4/group). The amounts of EV proteins isolated from Hep3B and primary hepatocyte cells treated with APAP alone or APAP+NAC were measured. *<i>P</i> < 0.05.</p

The amounts of total and liver-specific proteins in circulating EVs were increased in binge ethanol-exposed mice and alcoholics.

<p>(A) Wild-type male BALB/c mice (6 weeks old) were treated twice with saline (CON) or 6 g ethanol/kg via oral gavage at 12-h interval and were sacrificed 1 h after the second dose (n = 10/group). Representative H&E staining of formalin-fixed liver sections. (B) Protein amounts of the circulating EVs isolated from control or alcohol-exposed mice, as indicated. *<i>P</i> < 0.05. (C) Detection of the liver-specific proteins in circulating EVs from the indicated mouse plasma by immunoblot analysis. (D) Analysis of the protein amounts in circulating EVs isolated from the sera of healthy controls (n = 9), who never drank alcohol due to religious reasons, and alcoholics (n = 13) with hepatitis. (E) The amounts of liver-specific protein ALB in circulating EVs from the indicated groups were measured by ELISA for human ALB protein. *<i>P</i> < 0.05. (F) Detection of the liver-specific proteins in circulating EVs from the different groups by immunoblot analyses, as indicated. *<i>P</i> < 0.05.</p

Analysis of total protein amounts in circulating EVs from DGAL-, and TAA- treated mice.

<p>Wild-type male BALB/c mice (6 weeks old) were treated a single ip injection with PBS, DGAL (1,000 mg/kg), TAA (200 mg/kg), or PCN (2,400 mg/kg, control group) for 24 h (n = 10/group). (A) Representative H&E staining of formalin-fixed liver sections. (B) Plasma ALT level. (C) Protein amounts in circulating EVs were measured. (D) Detection of liver-specific proteins in circulating EVs by immunoblot analyses for each target protein. (E) The amounts of liver-specific mouse ALB in EVs from the indicated groups were measured by ELISA. *<i>P</i> < 0.05.</p

Clinical and biochemical characteristics of the study subjects.

<p>Clinical and biochemical characteristics of the study subjects.</p