Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)
Introduction
Although acetaminophen (APAP) is one of the most commonly used over-the-counter analgesic and antipyretic agents in the world, it accounts for more than 50% of all acute liver failure cases in the U.S. and Great Britain (Larson et al., 2005). For this reason, there is concern that has prompted extensive research aimed at elucidating the mechanism of APAP hepatotoxicity and how it may be prevented. Due to the similarities in injury and recovery between rodents and humans, rodent models have proven useful in studying signaling pathways involved in APAP hepatotoxicity (Park et al., 2005).
One experimental approach to modulate APAP hepatotoxicity in rodents is through auto/heteroprotection. Autoprotection is defined as resistance to toxicant re-exposure following acute, mild injury with the same toxicant, whereas heteroprotection is achieved when different toxicants are used for pretreatment and treatment. Carbon tetrachloride (CCl4) is one compound that has been used extensively as a chemical model of autoprotection. Mehendale and co-workers speculated in the early 1990s that compensatory hepatocellular proliferation is of critical importance to CCl4-induced autoprotection (Thakore and Mehendale, 1991). This hypothesis was supported by use of the antimitotic agent colchicine, which blocked autoprotection by preventing hepatocellular proliferation after the initial dose of CCl4, demonstrating that compensatory cell division following initial dosing with CCl4 is at least in part, responsible for the heightened tolerance and ability of the liver to recover from toxicant re-exposure (Rao and Mehendale, 1991).
Our laboratory has previously conducted studies using chemical treatments and conditions that reduce the severity of APAP toxicity in mice (Aleksunes et al., 2008a, Moffit et al., 2007b). Peroxisome proliferators such as clofibrate (CFB) are known to diminish APAP toxicity in mice (Manautou et al., 1994). Using knockout mice, we determined that protection by CFB is reversed in the absence of the PPARα receptor, demonstrating that its activation is necessary for hepatoprotection (Chen et al., 2000). Gene array analysis on livers from these rodents identified vanin-1 as a gene of interest. Vanin-1 (Vnn1) mRNA is significantly up-regulated in wild-type mice exhibiting protection from APAP toxicity, but not in PPARα-null mice (Moffit et al., 2007b). Increases in Vnn1 expression augment the levels of hepatic cystamine, which is an antioxidant capable of protecting against APAP hepatotoxicity (Miners et al., 1984, Moffit et al., 2007b). This increase in cystamine may explain why CFB protects the mouse liver from APAP toxicity. Vnn1 also modulates immune function by contributing to the extravasation of inflammatory cells to sites of injury (Meghari et al., 2007).
A mouse model of APAP autoprotection has been established in our laboratory to investigate the role and regulation of hepatobiliary drug transporters during development of resistance to APAP hepatotoxicity. We have determined that APAP autoprotection in mice is not due to differences in bioactivation or detoxification of APAP (Aleksunes et al., 2008a). These studies focused on the differential expression of members of the multidrug resistance-associated protein (Mrp) superfamily and their role in APAP autoprotection. These proteins are ATP-dependent membrane transporters responsible for the efflux of chemicals from the liver. mRNA and protein expression of the sinusoidal efflux transporter Mrp4 is elevated following APAP pretreatment, and its increased expression is localized to hepatocytes in centrilobular areas where compensatory cellular proliferation following pretreatment with mildly hepatotoxic doses of APAP is confined (Aleksunes et al., 2008a). Our studies also showed that colchicine treatment following administration of the priming dose of APAP reverses tolerance to hepatotoxicity, much like in the CCl4 model. The reversal in tolerance by colchicine is associated with a lack of induction in Mrp4 gene and protein expression that is usually seen with APAP. This suggests that Mrp4 expression is increased in proliferating hepatocytes as a mechanism for efflux of toxic by-products and to lower the chemical burden on hepatocytes, which in turn should lead to faster and more efficient recovery from APAP re-exposure (Aleksunes et al., 2008a).
While a role for Mrp4 in APAP autoprotection is well supported, we were interested in identifying other molecular pathways that might also contribute to the development of resistance to APAP hepatotoxicity resulting from pre-treatment to this toxicant. Therefore, C57BL/6J liver samples from our previous APAP autoprotection study (Aleksunes et al., 2008a) were subjected to gene array analysis. Statistically significant genes were analyzed individually and using the Causal Reasoning Engine (CRE) to gain further insight into the molecular mechanisms of autoprotection. CRE is a recently developed computational platform that provides hypotheses on the upstream molecular events that best explain gene expression profiles by interrogating prior biological knowledge (Enayetallah et al., 2011). Indeed, this approach did identify additional mechanisms that might further explain the molecular basis for autoprotection.
Section snippets
Chemicals
Acetaminophen, propylene glycol, and colchicine were purchased from Sigma-Aldrich (St Louis, MO). Zinc formalin was obtained from Fisher Scientific (Pittsburgh, PA). All other reagents were of reagent grade or better.
Animals
10–12-week old male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). Upon arrival, the mice were acclimated for one week. The mice were housed in a 12-h dark/light cycle in a temperature and humidity controlled environment. The mice were fed laboratory rodent
Results
APAP autoprotection in mice is a phenomenon that has been previously documented (Aleksunes et al., 2008a). In this model, mice are first pretreated with vehicle (controls) or 400 mg/kg APAP and then challenged 48 h later with a higher dose of APAP (600 mg/kg) or vehicle. The severity of APAP liver injury was assessed by plasma alanine aminotransferase (ALT) and histopathological analysis. In vehicle-pretreated mice, plasma ALT values at 24 h after APAP challenge were 1780 U/L whereas ALT values for
Discussion
Previous work in our laboratory indicates that induction of the liver efflux transporter multidrug resistance-associated protein 4 (Mrp4) and compensatory cellular proliferation after administration of mildly hepatotoxic doses of APAP are potentially involved in the development of tolerance to subsequent APAP administration in mice (Aleksunes et al., 2008a). However, it is possible that other factors also contribute to the development of tolerance to APAP toxicity. This would be consistent with
Conflict of interest statement
The authors do not have interests which conflict with publication of the data in this manuscript.
Acknowledgments
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases [DK069557, DK080774], the National Institute of Environmental Health Sciences [ES005022] and by Pfizer Inc.
References (52)
- et al.
Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2
Toxicol. Appl. Pharmacol.
(2008) - et al.
Temporal study of acetaminophen (APAP) and S-adenosyl-l-methionine (SAMe) effects on subcellular hepatic SAMe levels and methionine adenosyltransferase (MAT) expression and activity
Toxicol. Appl. Pharmacol.
(2010) - et al.
Flavin-containing monooxygenase-3: induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver
Toxicol. Appl. Pharmacol.
(2010) - et al.
Three soluble rat beta-galactoside-binding lectins
J. Biol. Chem.
(1985) - et al.
Cell-, tissue-, sex- and developmental stage-specific expression of mouse flavin-containing monooxygenases (Fmos)
Biochem. Pharmacol.
(2004) - et al.
Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism
Pharmacol. Ther.
(2005) - et al.
Protein disulfide isomerase in redox cell signaling and homeostasis
Free Radic. Biol. Med.
(2012) - et al.
Deficiency of the Nrf1 and Nrf2 transcription factors results in early embryonic lethality and severe oxidative stress
J. Biol. Chem.
(2003) - et al.
Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity
Toxicol. Appl. Pharmacol.
(1994) - et al.
Galectin-3 overexpression protects from cell damage and death by influencing mitochondrial homeostasis
FEBS Lett.
(2000)
Mechanism of action of paracetamol protective agents in mice in vivo
Biochem. Pharmacol.
Role of NAD(P)H:quinone oxidoreductase 1 in clofibrate-mediated hepatoprotection from acetaminophen
Toxicology
Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate
Toxicol. Appl. Pharmacol.
Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity
Biochem. Biophys. Res. Commun.
PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum
J. Biol. Chem.
Simultaneous induction of galectin-3 phosphorylated on tyrosine residue, p21(WAF1/Cip1/Sdi1), and the proliferating cell nuclear antigen at a distinctive period of repair of hepatocytes injured by CCl4
Biochem. Biophys. Res. Commun.
Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria. A role for synexin in galectin-3 translocation
J. Biol. Chem.
Emerging role of Nrf2 in protecting against hepatic and gastrointestinal disease
Toxicol. Pathol.
Coordinated expression of multidrug resistance-associated proteins (Mrps) in mouse liver during toxicant-induced injury
Toxicol. Sci.
Acquired resistance to acetaminophen hepatotoxicity is associated with induction of multidrug resistance-associated protein 4 (Mrp4) in proliferating hepatocytes
Toxicol. Sci.
Nrf2: a potential target for new therapeutics in liver disease
Clin. Pharmacol. Ther.
Interindividual differences of human flavin-containing monooxygenase 3: genetic polymorphisms and functional variation
Drug Metab. Dispos.
Human flavin-containing monooxygenases
Annu. Rev. Pharmacol. Toxicol.
An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen
Proc. Natl. Acad. Sci. U. S. A.
Peroxisome proliferator-activated receptor alpha-null mice lack resistance to acetaminophen hepatotoxicity following clofibrate exposure
Toxicol. Sci.
Causal reasoning on biological networks: interpreting transcriptional changes
Bioinformatics
Cited by (23)
Single-cell RNA sequencing reveals the dynamics of hepatic non-parenchymal cells in autoprotection against acetaminophen-induced hepatotoxicity
2023, Journal of Pharmaceutical AnalysisChronic copper treatment prevents the liver critical balance transcription response induced by acetaminophen
2019, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :The nuclear factors and nuclear receptors families HNF, FOX and PPAR, involved in hepatocyte development are the main regulators implicated in reversing liver failure in this system are also represented in our network controlling down-regulated genes [8]; supporting the hypothesis that acetaminophen induced a set of transcription factors previously identified with roles in modulating liver damage. In addition, nuclear factors (NRF) have been linked to a protective role during the adaptation process to the acetaminophen [40]. A three-year copper treatment in animals did not generate evidence of liver damage, while the copper treatment prevented a significant transcriptional response to acetaminophen challenge.
Idiosyncratic drug-induced liver injury in patients: Detection, severity assessment, and regulatory implications
2019, Advances in PharmacologyCitation Excerpt :Although the mechanisms underlying these elevations are often unclear, it is interesting that biomarker studies involving dosing of heparins (Harrill et al., 2012) and cholestyramine (Singhal et al., 2014) indicate that they result from hepatocyte injury or death (and not passive leakage, increased synthesis, or decreased clearance from the blood). The resolution of these injuries despite continued dosing presumably reflects engagement of compensatory mechanisms, and this has been shown with APAP in rodent studies (O'Connor, Koza-Taylor, Campion, et al., 2014). Dr. Robert Temple at the FDA has stated that drugs that are capable of causing IDILI also cause typically transient elevations in serum ALT > 3 × ULN in a small subset of treated patients (Temple, 2006).
Oxidative stress-responsive transcription factor NRF2 is not indispensable for the human hepatic Flavin-containing monooxygenase-3 (FMO3) gene expression in HepG2 cells
2016, Toxicology in VitroCitation Excerpt :The authors also showed that increased Fmo3 mRNA levels in response to 3-methylcholanthrene (3MC) and benzo(a)pyrene (BaP) treatment, but not by TCDD, in Hepa-1 cells was mediated by p53 binding to a response element in the Fmo3 promoter region (Celius et al., 2010). A gene array analysis performed in our laboratory also identified increased Fmo3 gene expression after APAP treatment (O'Connor et al., 2014). We have also demonstrated enhanced Fmo3 gene expression after treatment with other hepatotoxicants such as alpha-naphthyl isothiocyanate and after bile duct ligation (Rudraiah et al., 2014b; Rudraiah et al., 2014a).
Differential Fmo3 gene expression in various liver injury models involving hepatic oxidative stress in mice
2014, ToxicologyCitation Excerpt :Despite Fmo3 being considered non-inducible, studies with aryl hydrocarbon receptor (AhR) agonists in mice revealed liver Fmo3 gene induction (Celius et al., 2008, 2010). 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 autoprotected mice (Rudraiah et al., 2014).