Pregnane X Receptor (PXR) activation leads to liver steatosis

Of the originating work: Chander Negi, Lola Bajard, Jiri Kohoutek, and Ludek Blaha, Faculty of Science, Masaryk University, Brno, Czech Republic

Of the content populated in the AOP-Wiki:  John R. Frisch and Travis Karschnik, General Dynamics Information Technology, Duluth, Minnesota; Daniel L. Villeneuve, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN

Pregnane X receptor (PXR) belongs to a class of nuclear receptors [Aryl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GR), Liver X receptor (LXR), Peroxisome proliferator-activated receptor (PPAR), Retinoic acid receptor (RAR)] that are needed for normal liver function, but for which increased expression (i.e. activation by binding by chemical stressors) lead to liver injury, including steatosis (Mellor et al. 1996).  Pregnenolone and progesterone are ligands in normal molecular activation of PXR (Mellor et al. 1996), while an increasing number of chemical stressors have been shown to increase PXR expression (Bajard et al. 2019; Moya et al. 2020).  Activation of PXR has been linked to increased gene expression of CD36 (Zhou et al. 2006).  The transmembrane protein CD36 has been shown to have a central role in fatty acid influx (Glatz et al. 2010), with fatty acid influx one of the main pathways for increase in triglycerides in livers (Angrish et al. 2016).  Increases in triglycerides can result in histological changes to cell structure and disruption of normal biochemical function, ultimately resulting in steatosis as a primary adverse outcome (Angrish et al. 2016; Mellor et al. 1996).

This Adverse Outcome Pathway (AOP) was developed as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki.  The originating work for this AOP was: Negi, C.K., Bajard, L., Kohoutek, J., and Blaha, L.  2021.  An adverse outcome pathway based in vitro characterization of novel flame retardants-induced hepatic steatosis.  Environmental Pollution 289: 117855.  This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages. 

Flame retardants are of environmental and human health concern because of increased use and ability to leach into the environment.  Exposure concerns include effects on reproduction, development, neurology, and endocrine pathways (Negi et al. 2021).   This AOP focuses on a subset of endocrine disruption related to loss of lipid homeostasis, specifically the pathway in which activation of Pregnane X Receptor (PXR) leads to liver steatosis through increased fatty acid influx.  Environmental stressors result in activation of nuclear receptors linked to increases in triglyceride accumulation through several pathways.  One of the primary pathways linked to triglyceride accumulation, and focus of this AOP, is through activation of the PXR gene and coordinated molecular responses leading to increased fatty acid influx.  This pathway has been particularly well studied in mammals (humans, lab mice, lab rats).

The focus of the originating work was to use an AOP framework to integrate lines of evidence from multiple disciplines based on evolving guidance developed by the Organization for Economic Cooperation and Development (OECD).  Negi et al. (2021) provided initial network analysis based on empirical study of gene expression, lipid levels, and cell viability of human hepatocellular carcinoma cells (HepG2) exposed to 9 flame retardants (TDCIPP, TPHP, TMPP, TBBPA, EHDPP, TCEP, TNBP, TCIPP, and TBOEP) in order to obtain dose-response data.   ToxCast database analysis and in silico molecular modeling supplemented cell viability and lipid accumulation empirical data, and led to the proposed Adverse Outcome Pathway.

The originating authors conducted a literature search to develop a database of publications categorized by discipline or field of study: toxicology, epidemiology, exposure, and gene-environment interaction. The literature search relied on standard search engines such as Web of Science and Google Scholar, and the search strategy focused on toxicants known to disrupt lipid pathways in organisms, and diet studies with elevated levels of lipids. The originating authors reviewed references from individual citations to identify additional studies not captured through the literature search itself. They then included all relevant publications through 2023. Only studies focused primarily on developmental or neurotoxic endpoints were included; those focused on carcinogenesis or other systemic effects were not included unless there was a particular relevance to a neurotoxic or developmental outcome.

The scope of the aforementioned EPA project was limited to re-representing the AOP(s) as presented in the originating publication. The literature used to support this AOP and its constituent pages began with the originating publication and followed to the primary, secondary, and tertiary works cited therein. KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred.  

The authors of AOP 517 also referred to AOP 60: PXR activation to steatosis by Michelle Angrish and Brian Chorley.  In contrast to AOP 60, we have condensed the number of key events leading to increase in fatty acid influx.  We recognize that there is a complex interaction of genes within organisms, and focus attention on the role of upregulation of CD36 gene expression, leading to increased fatty acid influx.

Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N.  2016. Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis.  Toxicological Sciences 150(2): 261-268.

Bajard, L., Melymuk, L., and Blaha, L.  2019.  Prioritization of hazards of novel flame retardants using the mechanistic toxicology information from ToxCast and Adverse Outcome Pathways. Environmental Sciences Europe 31:14.

Glatz, J.F.C., Luiken, J.J.F.P., and Bonen, A.  2010.  Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism: Implications for Metabolic Disease.  Physiological Reviews 90: 367–417.

Mellor, C.L., Steinmetz, F.P., and Cronin, T.D.  2016.  The identification of nuclear receptors associated with hepatic steatosis to develop and extend adverse outcome pathways.  Critical Reviews in Toxicology, 46(2): 138-152.

Moya, M., Gomez-Lechon, M.J., Castell, J.V., and Jovera, R.  2010.  Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile.  Chemico-Biological Interactions 184: 376–387.

Negi, C.K., Bajard, L., Kohoutek, J., and Blaha, L.  2021.  An adverse outcome pathway based in vitro characterization of novel flame retardants-induced hepatic steatosis.  Environmental Pollution 289: 117855.

Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W.  2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway.  Journal of Biological Chemistry 281(21): 15013-15020.