Targeting the pregnane X receptor in liver injury.
ABSTRACT: The nuclear receptor pregnane X receptor (PXR) is a well-characterized hepatic xenobiotic sensor whose activation by chemically diverse compounds results in the induction of drug clearance pathways that rid the body of potentially toxic substances, thus conferring protection from foreign chemicals and endobiotics.PXR activities are implicated in drug-drug interactions and endocrine disruption. Recent evidence supports a hepatoprotective role for PXR in chronic liver injury, inhibiting liver inflammation through suppression of the NF-?B pathway. However, PXR-mediated induction of CYP3A enhances APAP-induced acute liver injury by generating toxic metabolites. While these observations implicate PXR as a therapeutic target for liver injury, they also caution against PXR activation by pharmaceutical drugs.While evidence of PXR involvement in acute and chronic liver injuries identifies it as a possible therapeutic target, it raises additional concerns for all drug candidates. The in vitro and in vivo tests for human PXR activation should be incorporated into the FDA regulations for therapeutic drug approval to identify potential liver toxicities. In addition, PXR pharmacogenetic studies will facilitate the prediction of patient-specific drug reactivities and associated liver disorders.
Project description:Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.
Project description:The liver plays a central role in transforming and clearing foreign substances. The continuous exposure of the liver to xenobiotics sometimes leads to impaired liver function, referred to as drug-induced liver injury (DILI). The pregnane X receptor (PXR) tightly regulates the expression of genes in the hepatic drug-clearance system and its undesired activation plays a role in DILI.This review focuses on the recent progress in understanding PXR-mediated DILI and highlights the efforts made to assess and manage PXR-mediated DILI during drug development.Future efforts are needed to further elucidate the mechanisms of PXR-mediated liver injury, including the epigenetic regulation and polymorphisms of PXR. Novel in vitro models containing functional PXR could improve our ability to predict and assess DILI during drug development. PXR inhibitors may provide chemical tools to validate the potential of PXR as a therapeutic target and to develop drugs to be used in the clinic to manage PXR-mediated DILI.
Project description:Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure and remains a critical problem in medicine. PARP1-dependent poly(ADPribosyl)ation is a key mediator of cellular stress responses and functions in multiple physiological and pathological processes. However, whether it is involved in the process of APAP metabolism remains elusive. In this study, we find that PARP1 is activated in mouse livers after APAP overdose. Pharmacological or genetic manipulations of PARP1 are sufficient to suppress the APAP-induced hepatic toxicity and injury, as well as reduced APAP metabolism. Mechanistically, we identify pregnane X receptor (PXR) as a substrate of PARP1-mediated poly(ADP-ribosyl)ation. The poly(ADP-ribosyl)ation of PXR in ligand-binding domain activates PXR competitively and solidly, facilitates its recruitment to target gene CYP3A11 promoter, and promotes CYP3A11 gene transcription, thus resulting in increases of APAP pro-toxic metabolism. Additionally, PXR silence antagonizes the effects of PARP1 on APAP-induced hepatotoxicity. These results identifies poly(ADP-ribosyl)ation of PXR by PARP1 as a key step in APAP-induced liver injury. We propose that inhibition of PARP1-dependent poly(ADP-ribosyl)ation might represent a novel approach for the treatment of drug-induced hepatotoxicity.
Project description:Hemorrhagic shock (HS) is a life-threatening condition associated with tissue hypoperfusion and often leads to injury of multiple organs including the liver. Pregnane X receptor (PXR) is a species-specific xenobiotic receptor that regulates the expression of drug-metabolizing enzymes (DMEs) such as the cytochrome P450 (CYP) 3A. Many clinical drugs, including those often prescribed to trauma patients, are known to activate PXR and induce CYP3A. The goal of this study is to determine whether PXR plays a role in the regulation of DMEs in the setting of HS and whether activation of PXR is beneficial or detrimental to HS-induced hepatic injury. PXR transgenic, knockout, and humanized mice were subject to HS, and the liver injury was assessed histologically and biochemically. The expression and/or activity of PXR and CYP3A were manipulated genetically or pharmacologically in order to determine their effects on HS-induced liver injury. Our results showed that genetic or pharmacological activation of PXR sensitized wild-type and hPXR/CYP3A4 humanized mice to HS-induced hepatic injury, whereas knockout of PXR protected mice from HS-induced liver injury. Mechanistically, the sensitizing effect of PXR activation was accounted for by PXR-responsive induction of CYP3A and increased oxidative stress in the liver. The sensitizing effect of PXR was attenuated by ablation or pharmacological inhibition of CYP3A, treatment with the antioxidant N-acetylcysteine amide, or treatment with a PXR antagonist. Conclusion: We have uncovered a function of PXR in HS-induced hepatic injury. Our results suggest that the unavoidable use of PXR-activating drugs in trauma patients has the potential to exacerbate HS-induced hepatic injury, which can be mitigated by the coadministration of antioxidative agents, CYP3A inhibitors, or PXR antagonists.
Project description:Overdose of acetaminophen (APAP), the active ingredient of Tylenol, is the leading cause of drug-induced acute liver failure in the United States. As such, it is necessary to develop novel strategies to prevent or manage APAP toxicity. In this report, we reveal a novel function of the liver X receptor (LXR) in preventing APAP-induced hepatotoxicity. Activation of LXR in transgenic (Tg) mice or by an LXR agonist conferred resistance to the hepatotoxicity of APAP, whereas the effect of LXR agonist on APAP toxicity was abolished in LXR-deficient mice. The increased APAP resistance in LXR Tg mice was associated with increased APAP clearance, increased APAP sulfation, and decreased formation of toxic APAP metabolites. The hepatoprotective effect of LXR may have resulted from the induction of antitoxic phase II conjugating enzymes, such as Gst and Sult2a1, as well as the suppression of protoxic phase I P450 enzymes, such as Cyp3a11 and Cyp2e1. Promoter analysis suggested the mouse Gst isoforms as novel transcriptional targets of LXR. The suppression of Cyp3a11 may be accounted for by the inhibitory effect of LXR on the PXR-responsive transactivation of Cyp3a11. The protective effect of LXR in preventing APAP toxicity is opposite to the sensitizing effect of pregnane X receptor, constitutive androstane receptor, and retinoid X receptor alpha.We conclude that LXR represents a potential therapeutic target for the prevention and treatment of Tylenol toxicity.
Project description:Liver injury after experimental acetaminophen treatment is mediated both by direct hepatocyte injury through a P450-generated toxic metabolite and indirectly by activated liver Kupffer cells and neutrophils. This study was designed to investigate the role of Notch signaling in the regulation of innate immune responses in acetaminophen (APAP)-induced liver injury. Using a mouse model of APAP-induced liver injury, wild-type (WT) and toll-like receptor 4 knockout (TLR4 KO) mice were injected intraperitoneally with APAP or PBS. Some animals were injected with ?-secretase inhibitor DAPT or DMSO vehicle. For the in vitro study, bone marrow-derived macrophages (BMMs) were transfected with Notch1 siRNA, TLR4 siRNA, and non-specific (NS) siRNA and stimulated with LPS. Indeed, paracetamol/acetaminophen-induced liver damage was worse after Notch blockade with DAPT in wild-type mice, which was accompanied by significantly increased ALT levels, diminished hairy and enhancer of split-1 (Hes1), and phosphorylated Stat3 and Akt but enhanced high mobility group box 1 (HMGB1), TLR4, NF-?B, and NLRP3 activation after APAP challenge. Mice receiving DAPT increased macrophage and neutrophil accumulation and hepatocellular apoptosis. However, TLR4 KO mice that received DAPT reduced APAP-induced liver damage and NF-?B, NLRP3, and cleaved caspase-1 activation. BMMs transfected with Notch1 siRNA reduced Hes1 and phosphorylated Stat3 and Akt but augmented HMGB1, TLR4, NF-?B, and NLRP3. Furthermore, TLR4 siRNA knockdown resulted in decreased NF-?B and NLRP3 and cleaved caspase-1 and IL-1? levels following LPS stimulation. These results demonstrate that Notch signaling regulates innate NLRP3 inflammasome activation through regulation of HMGB1/TLR4/NF-?B activation in APAP-induced liver injury. Our novel findings underscore the critical role of the Notch1-Hes1 signaling cascade in the regulation of innate immunity in APAP-triggered liver inflammation. This might imply a novel therapeutic potential for the drug-induced damage-associated lethal hepatitis.
Project description:Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC-MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXR? dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.
Project description:Efficient detoxification and clearance of cholesterol metabolites such as oxysterols, bile alcohols, and bile acids are critical for survival because they can promote liver and cardiovascular disease. We report here that loss of the nuclear xenobiotic receptor PXR (pregnane X receptor), a regulator of enterohepatic drug metabolism and clearance, results in an unexpected acute lethality associated with signs of severe hepatorenal failure when mice are fed with a diet that elicits accumulation of cholesterol and its metabolites. Induction of a distinct drug clearance program by a high-affinity ligand for the related nuclear receptor, the constitutive androstane receptor, does not overcome the lethality, indicating the unique requirement of PXR for detoxification. We propose that the PXR signaling pathway protects the body from toxic dietary cholesterol metabolites, and, by extension, PXR ligands may ameliorate human diseases such as cholestatic liver diseases and the associating acute renal failure.
Project description:Toxic liver injury is a leading cause of liver failure and death because of the organ's inability to regenerate amidst massive cell death, and few therapeutic options exist. The mechanisms coordinating damage protection and repair are poorly understood. Here, we show that S-nitrosothiols regulate liver growth during development and after injury in vivo; in zebrafish, nitric-oxide (NO) enhanced liver formation independently of cGMP-mediated vasoactive effects. After acetaminophen (APAP) exposure, inhibition of the enzymatic regulator S-nitrosoglutathione reductase (GSNOR) minimized toxic liver damage, increased cell proliferation, and improved survival through sustained activation of the cytoprotective Nrf2 pathway. Preclinical studies of APAP injury in GSNOR-deficient mice confirmed conservation of hepatoprotective properties of S-nitrosothiol signaling across vertebrates; a GSNOR-specific inhibitor improved liver histology and acted with the approved therapy N-acetylcysteine to expand the therapeutic time window and improve outcome. These studies demonstrate that GSNOR inhibitors will be beneficial therapeutic candidates for treating liver injury.
Project description:Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Inhibition of APAP metabolic activation and promotion in APAP disposition are important to protect against APAP-induced liver injury. Tumor suppressor p53 is traditionally recognized as a surveillance molecule to preserve genome integrity. Recent studies have emerged on discovering its functions in metabolic regulation. Our previous study reported that p53 promoted bile acid disposition and alleviated cholestastic syndrome. Here, we examined the effect of doxorubicin (Dox)-mediated p53 activation on APAP-induced hepatotoxicity in mice and revealed a novel role of p53 in regulating APAP metabolism and disposition. Histopathological and biochemical assessments demonstrated that administration of Dox (10?mg/kg/d) before APAP treatment (400?mg/kg) significantly alleviated APAP-induced hepatotoxicity. Dox treatment prevented APAP-induced GSH depletion and lipid peroxidation. p53-null mice were more susceptible to APAP-induced liver injury. Further, we found that the expression of drug-metabolizing enzymes and transporters CYPs, SULTs and MRPs was regulated by p53. Dox treatment also promoted Nrf2 activation and increased the expression of Nrf2 target genes including GST?/? and NQO1, which contribute to APAP detoxification. Overall, this study is the first to demonstrate the protective role of p53 in regulating APAP metabolism and disposition, which provides a potential new therapeutic target for APAP-induced liver injury.