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

Young-Eun Cho,Pyong-Gon Moon,Moon-Chang Baek,Esteban Mezey,Eun-Ju Im,Byoung-Joon Song,Jung-Eun Kim

doi:10.1371/journal.pone.0172463

Abstract

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 N-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.

Highlights

Extracellular vesicles (EVs), including exosomes (40–150 nm in diameter) and microvesicles (50–1,000 nm in diameter), are constantly released by most types of cells[1,2,3]
To evaluate the amounts of EV secreted from hepatocytes by APAP, we treated the rat primary culture hepatocytes with various concentrations of APAP, isolated the EVs from the culture media[31], and confirmed their size and purity by morphological assessment using transmission electron microscopy (TEM)
Immunoblot analysis showed that the EVs isolated from APAP-treated cells or saline control (CON) contained the well-established exosomal marker proteins such as CD9 and CD63 (Fig 1C)

Summary

Introduction

Extracellular vesicles (EVs), including exosomes (40–150 nm in diameter) and microvesicles (50–1,000 nm in diameter), are constantly released by most types of cells[1,2,3]. EVs contain cell-type-specific proteins, mRNA, and miRNA[4] and provide intercellular state information in various pathogenic processes. These informative EVs have been shown to be useful biomarkers for various diseases, including cancers[5]. The liver is a primary site of drug toxicity because it metabolizes exogenous compounds into reactive intermediates, which can cause acute liver failure[11]. The dosages of many drugs, most notably acetaminophen (APAP), are limited because of their potential to induce liver injury and acute failure. APAP-induced hepatotoxicity is a common consequence of APAP overdose that can cause acute liver failure and death especially in the presence of alcohol ingestion[13]

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