AOP ID and Title:

Short Title: PXR activation leads to liver steatosis

Authors

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

Status

Abstract

Pregnane X receptor (PXR) belongs to a class of nuclear receptors [Arhyl 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 decreased mitochondrial biochemical function or histological changes in mitochondria structure, ultimately resulting in steatosis as a primary adverse outcome (Angrish et al. 2016; Mellor et al. 1996).

Background

This Adverse Outcome Pathway (AOP) focuses on 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 particular well studied in mammals (humans, lab mice, lab rats).

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Overall Assessment of the AOP

1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship  between KEup and KEdown consistent with established biological knowledge?
Key Event Relationship (KER)	Level of Support   Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance.
Relationship 3100: Activation, Pregnane-X receptor, NR1l2 leads to Up Regulation, CD36	Strong support.  The relationship between activation of Pregnane-X receptor and Up Regulation of CD36 is broadly accepted and consistently supported across taxa.
Relationship 66: Up Regulation, CD36 leads to Increase, FA Influx	Strong support.  The relationship between Up Regulation of CD36 and Increase, FA Influx is broadly accepted and consistently supported across taxa.
Relationship 132: Increase, FA Influx leads to Accumulation, Triglyceride	Strong support. Increase, FA Influx is broadly recognized as a major pathway leading to accumulation of triglycerides, and consistently supported across taxa.
Relationship 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis	Strong support.  The relationship between accumulation of triglycerides and liver steatosis is broadly accepted and consistently supported across taxa.
Overall	Strong support.  Extensive understanding of the relationships between events from empirical studies from a variety of taxa, including frequent testing in lab mammals.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Life Stage: The life stage applicable to this AOP is all life stages with a liver.  Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.

Sex: This AOP applies to both males and females.

Taxonomic: This AOP appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

Essentiality of the Key Events

2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?
Key Event (KE)	Level of Support Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events. Moderate = Indirect evidence from experimental studies inferring essentiality of relationship between key events due to difficulty in directly measuring at least one of key events.
MIE 239: Activation, Pregnane-X receptor, NR1l2	Strong support.  Activation of Pregnane-X receptor is a primary activator for increases in CD36 gene expression.  Evidence is available from toxicant and gene-knockout studies.
KE 54 Up Regulation, CD36	Strong support.  Up Regulation of CD36 expression is one gene linked to increases in fatty acid influx.   Evidence is available from toxicant, gene-knockout, and high lipid diet studies.
KE 115 Increase, FA Influx	Moderate support.  Increase in fatty acid influx is a primary factor in increased triglyceride levels in cells.  Evidence is available from toxicant and gene-knockout studies.
KE 291 Accumulation, Triglyceride	Strong support. Accumulation of triglyceride is linked to liver steatosis.  Evidence is available from toxicant, gene-knockout, and high lipid diet studies.
AO 459 Increased, Liver Steatosis	Strong support. Liver steatosis occurs due to a variety of stressors and breakdown of multiple biochemical pathways and physiological changes with resulting increases in triglyceride levels.  Evidence is available from toxicant and high lipid diet studies.
Overall	Moderate to strong support.  Direct evidence from empirical studies from laboratory mammals for most key events, with more inferential evidence for fatty acid influx.

Weight of Evidence Summary

3. Empirical Support for Key Event Relationship: Does empirical evidence support that a  change in KEup leads to an appropriate change in KEdown?
Key Event Relationship (KER)	Level of Support  Strong =  Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.
Relationship 3100: Activation, Pregnane-X receptor, NR1l2 leads to Up Regulation, CD36	Strong support.  Increases in Pregnane X-receptor expression lead to increases in upregulation of CD36 expression, primarily from studies examining TOXCAST data, as well as changes in gene expression levels after exposure to chemical stressors.
Relationship 66: Up Regulation, CD36 leads to Increase, FA Influx	Strong support. Increases in upregulation of CD36 expression lead to increases in fatty acid influx, primarily through measured increases in CD36 gene expression and increased triglyceride levels.  Increased fatty influx is inferred from increased triglyceride levels rather than directly observed.
Relationship 132: Increase, FA Influx leads to Accumulation, Triglyceride	Strong support. Increases in fatty acid influx is recognized as a primary pathway to accumulation of triglycerides.
Relationship 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis	Strong support. Increases in accumulation of triglyceride is recognized as a primary pathway to liver steatosis.
Overall	Strong support. Exposure from empirical studies shows consistent change in both events from a variety of taxa, including frequent testing in lab mammals.

References

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.

Appendix 1

List of MIEs in this AOP

Event: 239: Activation, Pregnane-X receptor, NR1l2

Short Name: Activation, Pregnane-X receptor, NR1l2

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Life Stage: The life stage applicable to this AOP is all life stages with a liver.  Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.

Sex: This AOP applies to both males and females.

Taxonomic: This AOP appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

List of Key Events in the AOP

Event: 54: Up Regulation, CD36

Short Name: Up Regulation, CD36

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Key Event Description

Fatty acid translocase CD36 (FAT/CD36) is a scavenger protein mediating uptake and intracellular transport of long-chain fatty acids (FA) in diverse cell types ^[1], ^[2]. In addition, CD36 can bind a variety of molecules including acetylated low density lipoproteins (LDL), collagen and phospholipids ^[3]. CD36 has been shown to be expressed in liver tissue ^[4], ^[5]. It is located in lipid rafts and non-raft domains of the cellular plasma membrane and most likely facilitates LCFA transport by accumulating LCFA on the outer surface ^[6], ^[7], ^[8].

FAT/CD36 gene is a liver specific target of LXR activation ^[9]. Studies have confirmed that the lipogenic effect of LXR and activation of FAT/CD36 was not a simple association, since the effect of LXR agonists on increasing hepatic and circulating levels of triglycerides and free fatty acids (FFAs) was largely abolished in FAT/CD36 knockout mice suggesting that intact expression and/or activation of FAT/CD36 is required for the steatotic effect of LXR agonists ^[10], ^[11]. In addition to the well-defined pathogenic role of FAT/CD36 in hepatic steatosis in rodents the human up-regulation of the FAT/CD36 in NASH patients is confirmed ^[12]. There are now findings that can accelerate the translation of FAT/CD36 metabolic functions determined in rodents to humans ^[13] and suggest that the translocation of this fatty acid transporter to the plasma membrane of hepatocytes may contribute to liver fat accumulation in patients with NAFLD and HCV ^[14]. In addition, hepatic FAT/CD36 up-regulation is significantly associated with insulin resistance, hyperinsulinaemia and increased steatosis in patients with NASH and HCV G1 (Hepatitis C Virus Genotype1) with fatty liver. Recent data show that CD36 is also increased in the liver of morbidly obese patients and correlated to free FA levels ^[15].

References

1. ↑ Su & Abumrad 2009 - Su X., Abumrad N.A., Cellular fatty acid uptake: a pathway under construction. Trends
   Endocrinol. Metab., 20 (No 2), 72-77, 2009
2. ↑ He et al. 2011 - He J. et al, The emerging roles of fatty acid translocase/CD36 and the aryl hydrocarbon
   receptor in fatty liver disease, Exp. Med. And Biology, 236, 1116-1121, 2011
3. ↑ Krammer 2011 - Krammer J. et al, Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4
   and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells, Int. J. Med. Sci.,
   8(7), 599-614, 2011
4. ↑ Pohl et al. 2005 - Pohl J., et al, FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires
   plasma membrane rafts, Mol. Biol. Cell., 16 (No 1), 24-31, 2005
5. ↑ Cheung et al. 2007 - Cheung L., et al, Hormonal and nutritional regulation of alternative CD36 transcripts
   in rat liver--a role for growth hormone in alternative exon usage, BMC Mol. Biol., 8, 60,
   2007
6. ↑ Ehehalt et al. 2008 - Ehehalt R., et al, Uptake of long chain fatty acids is regulated by dynamic interaction
   of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts). BMC
   Cell. Biol., 9, 45, 2008
7. ↑ Pohl et al. 2005 - Pohl J., et al, FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires
   plasma membrane rafts, Mol. Biol. Cell., 16 (No 1), 24-31, 2005
8. ↑ Krammer 2011 - Krammer J. et al, Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4
   and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells, Int. J. Med. Sci.,
   8(7), 599-614, 2011
9. ↑ Zhou 2008 - Zhou J., Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and
   PPAR gamma in promoting steatosis, Gastroenterology, 134 (No 2),556-567, 2008
10. ↑ Febbraio et al. 1999 - Febbraio M., et al, A null mutation in murine CD36 reveals an important role in fatty
    acid and lipoprotein metabolism, J Biol Chem, 274, 19055–19062, 1999
11. ↑ Lee et al. 2008 - Febbraio M., et al, A null mutation in murine CD36 reveals an important role in fatty
    acid and lipoprotein metabolism, J Biol Chem, 274, 19055–19062, 1999
12. ↑ Zhu et al. 2011 - Zhu L., et al, Lipid in the livers of adolescents with non-alcoholic steatohepatitis:
    combined effects of pathways on steatosis, Metabolism Clinical and experimental, 30,
    1001-1011, 2011
13. ↑ Love-Gregory et al. 2011 - Love-Gregory L., Abumrad N.A., CD36 genetics and the metabolic complications of
    obesity, Current Opinions in Clinical Nutition and Metabolic Care, 14 (No 6), 527-534,
    2011
14. ↑ Miquilena-Colina et al. 2011 - Miquilena-Colina M.E., et al, Hepatic fatty acid translocase CD36 upregulation is
    associated with insulin resistance, hyperinsulinaemia and increased steatosis in nonalcoholic
    steatohepatitis and chronic hepatitis C, Gut., 60 (No 10), 1394-1402 , 2011
15. ↑ Bechmann et al. 2010 - Bechmann L.P., et al, Apoptosis is associated with CD36/fatty acid translocase
    upregulation in non-alcoholic steatohepatitis, Liver Int., 30 (No 6), 850-859, 2010

Event: 115: Increase, FA Influx

Short Name: Increase, FA Influx

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Key Event Description

Fat influx to the liver is usually increased under condition like obesity. Free fatty acids (FFA) increase in blood leads to an increase of FFA uptake in the liver. Especially the long chain fatty acids (LCFAs) are translocated across the plasma membrane, reassembled to triglycerides and stored in lipid droplets causing hepatic steatosis ^[1].

As mentioned above CD36 has consistently been shown to be expressed at the plasma membrane and to enhance LCFA uptake upon over-expression ^[2], ^[3].

References

1. ↑ Amacher 2011 - Amacher D.E., The mechanistic basis for the induction of hepatic steatosis by
   xenobiotics, Expert Opinion on Drug Metabolism and Toxicology, 7 (No 8), 949-965,
   2011
2. ↑ Baranowski 2008 - Baranowski, Biological role of liver X receptors, Journal of Physiology and
   Pharmacology, 59 (Suppl 7), 31–55, 2008
3. ↑ Su & Abumrad 2009 - Su X., Abumrad N.A., Cellular fatty acid uptake: a pathway under construction. Trends
   Endocrinol. Metab., 20 (No 2), 72-77, 2009

Event: 291: Accumulation, Triglyceride

Short Name: Accumulation, Triglyceride

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Key Event Description

Leads to Fatty Liver Cells.

List of Adverse Outcomes in this AOP

Event: 459: Increased, Liver Steatosis

Short Name: Increased, Liver Steatosis

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa.

Key Event Description

Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes.

Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes.

Role in biology: steatosis is an adverse endpoint. 

 

Description from EU-ToxRisk:

Activation of stellate cells results in collagen accumulation and change in extracellular matrix composition in the liver causing fibrosis. (Landesmann, 2016)(Koo et al 2016)

How it is Measured or Detected

Steatosis is measured by lipidomics approaches that measure lipid levels, or by histology.

Regulatory Significance of the AO

Steatosis is a regulatory endpoint and has been used as an endpoint in many US EPA assessments, including IRIS assessments.

References

Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2).

https://doi.org/10.1016/j.molcel.2005.08.010

Koo, J. H., Lee, H. J., Kim, W., & Kim, S. G. (2016). Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2. Gastroenterology, 150(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039

Appendix 2

List of Key Event Relationships in the AOP