Date of Completion

6-13-2013

Embargo Period

6-13-2013

Keywords

Vanin-1, Pantetheinase, Cysteamine, Acetaminophen, Hepatotoxicity

Major Advisor

Dr. Jose Manautou

Associate Advisor

Dr. Michael Lawton

Associate Advisor

Dr. Theodore Rasmussen

Associate Advisor

Dr. Angela Slitt

Field of Study

Pharmaceutical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Pretreatment with peroxisome proliferators (PPs) is protective in rodent models of acetaminophen (APAP)-induced hepatotoxicity. Protection is associated with an induction of vascular non-inflammatory molecule 1 (Vanin-1; Vnn1) gene expression in liver. This thesis investigates whether Vnn1 expression is protective against APAP-induced toxicity and whether Vnn1 upregulation is involved in the PP-mediated mechanism of protection.

Mice lacking Vnn1 are more susceptible to APAP hepatotoxicity despite no apparent differences in APAP bioactivation or detoxification pathways. Enhanced susceptibility to APAP in Vnn1 knockout mice is associated with a deficiency in compensatory immune cell infiltration and hepatocellular repair in and around areas of hepatic centrilobular necrosis 48 hours after APAP treatment. Gene induction of cytokines involved with pro-inflammatory M1 stimulation is also deficient in the livers of Vnn1 knockout mice at the same time point.

A Vnn1 overexpression construct was stably transfected into the human hepatocyte cell line HC04. Exposure of HC04-VN1 cells to APAP treatment resulted in modest cytoprotection compared to HC04-EV cells as determined by the leakage of lactate dehydrogenase into media. Together, the data indicate that Vnn1 expression is protective against APAP-induced toxicity both in vitro and in vivo.

An in vitro model for investigating the mechanism of PP-mediated protection against APAP is also described. HC04 cells exhibit time- and dose-dependent increases in cytotoxicity following APAP exposure. Treatment with Wy-14,643 results in the induction of peroxisome proliferator activated receptor alpha (PPARα)-responsive genes acyl CoA oxidase 1 (Acox1), adipose differentiation related protein (Adrp) and Vnn1. Pretreatment with Wy-14,643 is partially protective in HC04 cells following APAP-induced cytotoxicity, though neither Vnn1 protein nor enzymatic activity are enhanced in whole cell lysates. Together, the studies suggest that enhanced Vnn1 activity is not involved with the observed partial protection by Wy-14,643 in HC04 monocultures.

Together, these investigations provide evidence that Vnn1 expression modulates APAP toxicity both in vivo and in vitro. Furthermore, the HC04-VN1 and PP-treated HC04 cell models described here represent two potential systems in investigating protective mechanisms against APAP-induced toxicity.

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