Date of Completion

8-4-2014

Embargo Period

8-4-2014

Keywords

Acetaminophen, FMO3, Autoprotection, Hepatotoxicity

Major Advisor

Dr. José E. Manautou,

Associate Advisor

Dr. Ronald N. Hines

Associate Advisor

Dr. Urs A. Boelsterli

Associate Advisor

Dr. Richard S. Bruno

Field of Study

Pharmaceutical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Mice pretreated with a mild toxic dose of acetaminophen (APAP) acquire resistance to a second, higher APAP dose. This phenomenon is termed APAP autoprotection and the exact mechanism by which such resistance develops is not clearly known. Given the prevalence of APAP-hepatotoxicity and the human health impact of this potentially hepatotoxic agent, a further understanding of the mechanism(s) involved in such protection are of considerable significance and could lead to new modalities of treatment of acute drug-induced liver injury. The work presented in this thesis investigates FMO3 gene expression during APAP-induced liver injury as well as the functional significance of FMO3 over-expression during APAP-induced liver injury. Furthermore, FMO3 gene regulation during oxidative stress conditions is also examined.

Acetaminophen treatment resulted in up-regulation of liver Fmo3 protein in male mice. Female mice express higher liver Fmo3 than males and are highly resistant to APAP hepatotoxicity. Inhibition of Fmo3, by methimazole, renders female mice susceptible to APAP-induced liver injury. These findings are suggestive of a protective function for Fmo3. In addition to APAP, ANIT and BDL also increase Fmo3 gene expression in mice. Because these hepatotoxicants also induce oxidative stress, we also investigated the potential role of the oxidative stress sensor and transcription factor, NRF2, in FMO3 gene regulation. Both in vivo and in vitro results show that transcriptional regulation of FMO3 might not involve the NRF2-KEAP1 regulatory pathway.

Human liver can adapt to APAP-induced hepatotoxicity similar to our APAP autoprotection mouse model. The last part of this dissertation examined FMO3 gene induction in a human hepatoma cell line, HepaRG. APAP induced FMO3 gene expression in HepaRG cells, and over-expression of FMO3 protects cells against APAP-induced cytotoxicity. The unexpected observation of a faster differentiation phenotype in HepaRG cells over-expressing FMO3 suggests that FMO3 may play an important role in cellular differentiation.

Collectively, data presented in this thesis provide evidence for Fmo3 as a novel genetic determinant of APAP-induced liver injury. Furthermore, this thesis describes a novel protective function for FMO3. Findings from HepaRG cells suggest that FMO3 over-expression in response to APAP may be a driving force for differentiation in regenerating hepatocytes.

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