We have been studying the mechanism of chemically-induced liver injury for many years. Alpha-naphthyl isothiocyanate (ANIT) is a common hepatotoxicant experimentally used to reproduce the pathologies of drug-induced liver injury in humans, but the mechanism of its toxicity remains unclear. To determine the metabolic alterations following ANIT exposure, metabolomics was performed by use of liquid chromatography-mass spectrometry. 1-Stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:0) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:1) were significantly increased in serum from ANIT-treated mice, and this increase resulted from altered expression of genes encoding the lipid metabolism enzymes Chka and Scd1. ANIT also increased NF-kB/IL-6/STAT3 signaling, and in vitro luciferase reporter gene assays revealed that LPC 18:0 and LPC 18:1 could activate NF-kB in a concentration-dependent manner. Activation of the ligand-activated nuclear receptors peroxisome proliferator-activated receptor (PPAR)alpha by Wy-14,643 (0.1%) reduced ANIT-induced liver injury, as indicated by lowered serum levels of liver-derived enzymes ALT and AST, and liver histology. This study demonstrated a role for the lipid-regulated NF-kB/IL-6/STAT3 axis in ANIT-induced hepatotoxicity, and suggests that PPARalpha may be a potential therapeutic target for the prevention of drug-induced cholestatic liver injury. In another study, analysis of liver, serum, bile, ileum, and cecum of vehicle- and ANIT-treated mice revealed significant alterations of bile acid metabolites, including increased tauroursodeoxycholic acid, taurohydrodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid, and decreased omega-, beta- and T-alpha/beta-murideoxycholic acid, cholic acid, and taurocholic acid in the ANIT-treated groups. In accordance with these changes, ANIT treatment altered the expression of mRNAs encoded by genes responsible for the metabolism and transport of bile acids and cholesterol. This modulation of bile acid metabolis had not previously been appreciated and its role in ANIT-induced liver injury is not known. Glycyrrhizin (GL), the major pentacyclic triterpene isolated from licorice, is known for its detoxifying and hepatoprotective properties, as well as many other pharmacological activities. GL is hydrolyzed by glucuronidase expressed by intestinal bacteria to its active principle aglycone, 18beta-glycyrrhetinic acid (GA). GL and GA possess several pharmacological activities, such as anti-inflammatory, hepatoprotective, anti-tumor, and anti-viral. GL and GA have been used for more than 20 years in the treatment and prevention of hepatitis, chronic bronchitis, gastritis, tumor growth and immunological disorders. The known anti-inflammatory and hepatoprotective effects of GL and GA suggested their potential use as a therapy for chemically-induced cholestasis such as that mediated by ANIT. Pre-treatment with GL and GA prevented ANIT-induced liver damage and reversed the alteration of bile acid metabolites and the mRNAs encoding of bile acid transport and metabolism proteins. These results suggested that GL/GA could prevent drug-induced liver injury and ensuing disruption of bile acid metabolism in humans. In another study, we investigated the metabolism of the commonly used herbicide flurochloridone (FLC) to determine if it is metabolized to a form that could cause genetic and cellular damage and toxicity. Agrochemicals, including pesticides and fertilizers, are used to increase the agricultural productivity and crop yields. However, their pervasive use in farming can result in a negative impact on the environment. The prevalent exposure of the populations to these substances has caused concerns over their potential health consequences. FLC is a herbicide used world-wide that is thought to be safe. FLC belongs to the phytoene desaturase bleaching herbicide family, and is a selective herbicide that is widely applied, especially in Asia and North America, in the control of many broadleaf weeds and annual grasses in cereal, sunflower, and potato crops, among others. FLC is absorbed by roots and stems and causing bleaching of the leaves through interference with carotenoid biosynthesis. However, due to its potential genotoxicity, cytotoxicity, and even systematic toxicity, there are increasing concerns about human exposure to this compound. Thus, the metabolism and bioactivation of FLC was investigated. After oral administration to mice, 27 metabolites were identified by ultra-high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry and with further structural identification with nuclear magnetic resonance spectroscopy. Hydroxylation and oxidative dechlorination were the major phase I pathways, while glutathione (GSH) and N-acetylcysteine conjugations were two major phase II pathways, indicating the formation of reactive intermediate. In vitro microsomal and cytosolic studies revealed that a GSH conjugate (M13) was the predominant metabolite of FLC formed through a nucleophilic SN2 substitution of 3-Cl by GSH; this pathway NADPH independent and accelerated by glutathione S-transferase (GST). Further, a kinetic study showed that M13 formation in both human liver microsomes and cytosols obeyed typical Michaelis-Menten kinetics. The maximum clearance (Vmax/Km) of GSH conjugation in human liver microsomes was approximately 5.5-fold higher than human liver cytosol, thus implying that microsomal GST was mainly responsible for M13 formation. These findings are important for understanding the potential hazard of human exposure to FLC and the potential effects on wild-life.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005708-26
Application #
9556206
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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