Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

Abstract

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 hr later with 600 mg APAP/kg. Livers were obtained 4 or 24 hr later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430_2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection.

Differential Fmo3 gene expression in various liver injury models involving hepatic oxidative stress in mice

“…A recent gene array analysis performed in our laboratory also demonstrated Fmo3 gene induction in the APAP autoprotection mouse model (mice receiving a low hepatotoxic APAP dose that become resistant to a subsequent higher APAP dose)(O'Connor et al, 2014). Unlike with AhR agonists that result in marginal increases in Fmo3 protein expression in mouse liver, we showed significant increases in Fmo3 protein levels by 15-fold in APAP autoprotectedmice(Rudraiah et al, 2014).…”

From hepatoprotection models to new therapeutic modalities for treating liver diseases: a personal perspective

“…This observation is in agreement with a recent finding by Leiser et al , who demonstrated that FMO enzymes increase the lifespan and enhance proteostasis in nematodes undergoing a hypoxic response, which is another form of stress/cytotoxicity 31 . It is intriguing that FMO3, once considered to be non-inducible by xenobiotic treatment, has been shown not only to be inducible by our group and others 12, 13, 32, 33 but also to protect against APAP-induced cytotoxicity in hepatocytes 13 . This highlights the evolving nature of our understanding about FMO3 function, which calls for more mechanistic studies to delineate FMO3’s role in protecting against APAP hepatotoxicity.…”

“…It is intriguing and worth noting that a similar shift in localization of flavin-containing monooxygenase-3 (FMO3) was also noted in livers of our APAP autoprotection model 13 . Fmo3 was identified from a microarray study as a potential gene contributing to resistance to liver toxicity in our APAP autoprotection mouse model 12 . Similar to CYP450s, FMO3 is a phase 1 drug-metabolizing enzyme involved in the oxygenation of nitrogen- and sulfur-containing endogenous substrates (such as cysteamine, trimethylamine, etc.)…”

“…With the advent of sophisticated techniques that allow comprehensive, global evaluation of differential gene and protein expression, we have adopted gene array analysis approaches to address mechanistic toxicology questions in both heteroprotection and autoprotection models 12, 15 . More recently, Eakins et al also addressed this subject through proteomic analysis in another rodent model of APAP autoprotection involving repeated APAP administration every 24 hours for up to 96 hours 14 .…”

“…The gene array analysis performed by our group in our APAP autoprotection model identified many differentially expressed genes that could contribute to the development of resistance to APAP hepatotoxicity upon re-exposure to this toxicant. Further analysis of differentially expressed genes using a computational platform known as Causal Reasoning Engine (CRE) provided insights into candidate signaling pathways mediating autoprotection 12 . CRE works by interrogating prior biological knowledge on the observed gene expression profile and provides hypotheses on the upstream molecular events that best explain the expression changes observed in the dataset 28 .…”