Activation, LXR

117-81-7

BJQHLKABXJIVAM-UHFFFAOYNA-N

BJQHLKABXJIVAM-UHFFFAOYSA-N

Di(2-ethylhexyl) phthalate

1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester DEHP 1,2-Benzedicarboxylic acid, bis(2-ethyl-hexyl) ester 1,2-Benzenedicarboxylic acid bis(2-ethylhexyl) ester 1,2-Benzenedicarboxylic acid, 1,2-bis(2-ethylhexyl) ester 1,2-Benzenedicarboxylic acid,bis(2-ethylhexylester) Bis(2-ethylhexyl) 1,2-benzenedicarboxylate Bis(2-ethylhexyl) o-phthalate bis(2-ethylhexyl) phthalate Bis(2-ethylhexyl)phthalat Bis(2-ethylhexyl)phthalate Bisoflex 81 Bisoflex DOP Corflex 400 Di(2-ethylhexyl)phthalate Di(isooctyl) phthalate Di-2-ethylhexlphthalate Di-2-ethylhexyl phthalate DI-2-ETHYLHEXYL-PHTHALATE Diacizer DOP Diethylhexyl phthalate Dioctylphthalate DOF Ergoplast FDO Ergoplast FDO-S ETHYLHEXYL PHTHALATE Eviplast 80 Eviplast 81 Fleximel Flexol DOD Flexol DOP ftlalato de bis(2-etilhexilo) Garbeflex DOP-D 40 Good-rite GP 264 Hatco DOP Jayflex DOP Kodaflex DEHP Kodaflex DOP Monocizer DOP NSC 17069 Palatinol AH Palatinol AH-L Phtalate de Bis (Ethyle-2-Hexyle) Phtalate de bis(2-ethylhexyle) PHTHALATE, BIS(2-ETHYLHEXYL) Phthalic acid di(2-ethylhexyl) ester Phthalic acid, bis(2-ethylhexyl) ester PHTHALIC ACID, BIS(2-ETHYLHEXYL)ESTER PHTHALSAEURE-BIS-(2-AETHYLHEXYL)-ESTER Pittsburgh PX 138 Plasthall DOP Reomol D 79P Sansocizer DOP Sansocizer R 8000 Sconamoll DOP Staflex DOP Truflex DOP Vestinol AH Vinycizer 80 Vinycizer 80K Witcizer 312

DTXSID5020607

97322-87-7

GXPHKUHSUJUWKP-UHFFFAOYNA-N

GXPHKUHSUJUWKP-UHFFFAOYSA-N

Troglitazone

DTXSID8023719

688-73-3

DBGVGMSCBYYSLD-UHFFFAOYSA-N

Tributyltin

hidruro de tri-n-butilestano Hydridotris(butyl)tin Hydrure de tributylstannane hydrure de tri-n-butyletain Tributylstannane Tributylstannic hydride Tributylstannyl hydride TRIBUTYLTIN HYDRIDE Tri-n-butylstannane tri-n-butyltin hydride Tri-n-butylzinnhydrid

DTXSID0040709

7440-61-1

JFALSRSLKYAFGM-UHFFFAOYSA-N

Uranium

Uranium, isotope of mass 238 238U Element UN 2979 (DOT) Uranium I

DTXSID1042522

7440-43-9

BDOSMKKIYDKNTQ-UHFFFAOYSA-N

Cadmium

Cadimium CADMIUM BLUE CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER

DTXSID1023940

PR:000011394

PR oxysterols receptor LXR-beta

PR:000011395

PR oxysterols receptor LXR-alpha

PR:000001905

PR platelet glycoprotein 4

CHEBI:35366

CHEBI fatty acid

PR:000010460

PR carbohydrate-responsive element-binding protein

PR:000015611

PR sterol regulatory element-binding protein 1

PR:000007348

PR fatty acid synthase

PR:000014497

PR acyl-CoA desaturase

CHEBI:17855

CHEBI triglyceride

D005234

MESH fatty liver

PR:000013058

PR peroxisome proliferator-activated receptor gamma

GO:0005739

GO mitochondrion

GO:0023052

GO signaling

GO:0010467

GO gene expression

GO:2000193

GO positive regulation of fatty acid transport

GO:0032933

GO SREBP signaling pathway

GO:0004312

GO fatty acid synthase activity

GO:0006633

GO fatty acid biosynthetic process

GO:0035357

GO peroxisome proliferator activated receptor signaling pathway

WIKI increased

WIKI occurrence

WIKI functional change

Mono(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Di(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Troglitazone

2016-11-29T18:42:26

Tributyltin

2017-07-24T16:32:02

Uranium

2021-08-05T14:28:50

Nanoparticles and Micrometer Particles

2022-02-04T13:43:43

Cadmium

2017-10-25T08:33:12

WikiUser_28

Vertebrates

10095

NCBI mice

WCS_9606

common toxicological species human

10116

NCBI rat

10090

NCBI mouse

WCS_7227

common ecological species Drosophila melanogaster

6239

NCBI Caenorhabditis elegans

10090

NCBI Mus musculus

Activation, LXR

Molecular

<h3>The LXR receptor</h3> Liver X receptors (LXR) are ligand-activated transcription factors of the nuclear receptor superfamily first identified in 1994 in rat liver (Apfel et al. 1994, Song 1994). There are two LXR isoforms termed a and ß (NR1H3 and NR1H2) which upon activation form heterodimers with retinoid X receptor (RXR) and bind to the LXR response element found in the promoter region of the target genes (Baranowski 2008). LXRs were shown to function as sterol sensors protecting the cells from cholesterol overload by stimulating reverse cholesterol transport and activating its conversion to bile acids in the liver (Baranowski 2008). LXRa expression is restricted to liver, kidney, intestine, fat tissue, macrophages, lung, and spleen and is highest in liver, hence the name liver X receptor a (LXRa). LXRβ is expressed in almost all tissues and organs, hence the early name UR (ubiquitous receptor) (Ory 2004). The different pattern of expression suggests that LXRa and LXRβ have different roles in regulating physiological function. This is also supported from the observation that LXRa deficient mice do not develop hepatic steatosis when treated with LXR agonist that activates both types (Lund et al. 2006) and consequently the role of the two isoforms in relation to adverse effects could be different.   <h3>The molecular initiating event</h3> Generally speaking chemicals that are able to act through NRs are usually specific ligands. These chemicals are mainly lipophilic and they mimic the action of natural hormones. However, in some cases hydrophilic chemicals (like phthalates) are also capable to act as ligands in NRs due to the molecular structure of the proteins and the pocket sites of the receptors. The molecular initiating event in the presented MoA is the binding to the LXR or the permissive RXR of the LXR-RXR dimer leading to activation. LXR activation can be achieved via a wide range of endogenous neutral and acidic ligands as shown by crystallographic analysis (Williams et al. 2003). There are known endogenous but also synthetic ligands that can act as agonists. Endogenous agonists for this receptor are the oxysterols (oxidized cholesterol derivatives like 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid) mainly with similar affinity for the two isoforms (Baranowski 2008). Oxysterols bind directly to the typical hydrophobic pocket in the C-terminal domain (Williams et al. 2003). Other endogenous ligands are the D-glucose and D-Glucose-6-phosphate (Mitro 2007). However, the hydrophilic nature of glucose and its low affinity for LXR present a challenge to the central dogma about the nature of the NR-ligand interaction (Lazar & Wilson 2007). Unsaturated fatty acids have also been shown to bind and regulate LXRa activity in cells. However, in contrast to the role of oxysterols, the biological relevance of this observation has not been established in vivo (Pawar et al. 2003). The function of LXRs is also modulated by many currently used drugs such as statins, fibrates, and thazolidinedione derivatives (Jamroz-Wiśniewska et al. 2007). Some synthetic LXR agonists have been developed like the non-steroidal agonists T0901317 and GW3965 (Schultz et al 2000, Collins et al. 2002). LXR forms a permissive dimer with the RXR which means that chemicals that can activate this receptor can trigger the same pathway as the LXR agonists. The endogenous RXR agonist is 9-cis-retinoic acid (Heyman et al. 1992) while synthetic agonists include LGD1069 and LG100268 (Boehm et al. 1994 and 1995). In addition to the agonist binding in the LXR there are other mechanisms for its control. LXRa gene promoter contains also functional peroxisome proliferator response element (PPRE) and peroxisome proliferator-activated receptor (PPAR) a and γ agonists were shown to stimulate LXRa expression in human and rodent (Baranowski 2008). Control of the LXRa expression is also dependent on insulin and post-translationally by protein kinase A that phosphorylates receptor protein at two sites thereby impairing its dimerization and DNA-binding (Baranowski 2008).   <h3>Identification of the site of action</h3> As already mentioned above LXR isoforms are expressed in various tissues but in relation to the presented MoA we refer to LXRs that are expressed in the hepatocytes. Nuclear receptors may be classified into two broad classes according to their sub-cellular distribution in the absence of ligand. Type I NRs (like ER and AhR) are located in the cytosol (and they are translocated into the nucleus after ligand binding) while type II NRs like LXRs (but also PXR, PPARa and PPARγ) are located in the nucleus of the cell. The specific site of binding and the affinity of a ligand for the LXRs depend on the structure of the ligand.   <h3>Binding in the LXREs and target genes transcription</h3> Upon ligand-induced activation both isoforms form obligate heterodimers with the retinoid X receptor (RXR) and regulate gene expression through binding to LXR response elements (LXREs) in the promoter regions of the target genes (Fig. 1). The LXRE consists of two idealized hexanucleotide sequences (AGGTCA) separated by four bases (DR-4 element). <a class="image" href="/wiki/index.php/File:Mechanism-of-transcriptional-regulation-mediated-by-LXRs.png"><img alt="Mechanism-of-transcriptional-regulation-mediated-by-LXRs.png" src="/wiki/images/b/b0/Mechanism-of-transcriptional-regulation-mediated-by-LXRs.png" style="height:350px; width:449px" /></a> Figure 1. Mechanism of transcriptional regulation mediated by LXRs. RXR - retinoid X receptor, LXRE - LXR response element (Baranowski 2008) Target genes of LXRs are involved in cholesterol and lipid metabolism regulation (<a href="#cite_note-1">[1]</a>, <a href="#cite_note-2">[2]</a>) including: <ul> <li>ABC - ATP Binding Cassette transporter isoforms A1, G1, G5, and G8</li> <li>ApoE - Apolipoprotein E</li> <li>CETP - Cholesterylester Transfer Protein</li> <li>CYP7A1 - Cytochrome P450 isoform 7A1 - cholesterol 7a-hydroxylase</li> <li>FAS - Fatty Acid Synthase</li> <li>LPL - Lipoprotein Lipase</li> <li>LXR-a - Liver X Receptor-a</li> <li>SREBP-1c - Sterol Response Element Binding Protein 1c</li> <li>ChREBP - Carbohydrate Response Element Binding Protein</li> <li>FAT/CD36 – Fatty acid uptake transporter (liver)</li> </ul>   <h3>Auto-regulation of the LXRa</h3> Human specific auto-regulated expression specifically of the LXRa has been demonstrated from several studies (Laffitte et al. 2001, Whitney et al. 2001, Li et al. 2002, Kase et al. 2007). Human LXRa gene promoter has a functional LXRE activated by both LXRa and β. In addition human liver LXRa expression is induced by both natural and synthetic LXR agonists.

Liver X receptor (LXR) activation is measured by changes in gene expression and protein levels.  Effects of LXR on expression of downstream genes can be investigating using metabolomics and RT-qPCR approaches.  In addition, targeted ToxCast assays using SeqAPASS evaluations can evaluate gene expression changes from chemical exposure for model species (e.g. Lalone et al. 2018).  Relevent ToxCast assays are ATG_LXRa_TRANS; ATG_LXRb_TRANS; ATG_DR4_LXR_CIS (U.S. EPA 2024).

Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to increased opportunity to upregulate gene expression. Sex: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

CL:0000182

CL hepatocyte

High Unspecific

High

Adult

Moderate

Juvenile

High

<ol> <li><a href="#cite_ref-1">↑</a> Peet 1998 - Peet D.J., Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the Nuclear Oxysterol Receptor LXRa in mammals, Cell, 93, 693–704, 1998</li> <li><a href="#cite_ref-2">↑</a> Edwardsa et al. 2002 - Edwardsa P.A., et al, LXRs; Oxysterol-activated nuclear receptors that regulate genes controlling lipid homeostasis, (Oxidized Lipids as Potential Mediators of Atherosclerosis), Vascular Pharmacology, 38 (No 4), 249–256, 2002</li> <li>LaLone, C.A., Villeneuve, D.L., Doering, J.A., Blackwell, B.R., Transue, T.R., Simmons, C.W., Swintek, J., Degitz, S.J., Williams, A.J., and Ankley, G.T.  2018.  Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results.  Environmental Science and Technology 52: 13960−13971.</li> <li>U.S. EPA. 2024. ToxCast & Tox21 Summary Files from invitrodb_v4. Retrieved from https://www.epa.gov/chemical-research/toxicity-forecaster-toxcasttm-data. </li> </ol> NOTE: Italics symbolize edits from John Frisch AOPWiki 2016-11-29T18:41:23

2024-05-21T10:33:01

Up Regulation, CD36

Molecular

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 <a href="#cite_note-1">[1]</a>, <a href="#cite_note-2">[2]</a>. In addition, CD36 can bind a variety of molecules including acetylated low density lipoproteins (LDL), collagen and phospholipids <a href="#cite_note-3">[3]</a>. CD36 has been shown to be expressed in liver tissue <a href="#cite_note-4">[4]</a>, <a href="#cite_note-5">[5]</a>. 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 <a href="#cite_note-6">[6]</a>, <a href="#cite_note-7">[7]</a>, <a href="#cite_note-8">[8]</a>. FAT/CD36 gene is a liver specific target of LXR activation <a href="#cite_note-9">[9]</a>. 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 <a href="#cite_note-10">[10]</a>, <a href="#cite_note-11">[11]</a>. 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 <a href="#cite_note-12">[12]</a>. There are now findings that can accelerate the translation of FAT/CD36 metabolic functions determined in rodents to humans <a href="#cite_note-13">[13]</a> 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 <a href="#cite_note-14">[14]</a>. 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 <a href="#cite_note-15">[15]</a>.

CD36 is measured by changes in gene expression and protein levels.

Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to increased opportunity to upregulate gene expression. Sex: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

CL:0000182

CL hepatocyte

High Unspecific

High

Adult

Moderate

Juvenile

High

<ol> <li><a href="#cite_ref-1">↑</a> 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</li> <li><a href="#cite_ref-2">↑</a> 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</li> <li><a href="#cite_ref-3">↑</a> 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</li> <li><a href="#cite_ref-4">↑</a> 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</li> <li><a href="#cite_ref-5">↑</a> 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</li> <li><a href="#cite_ref-6">↑</a> 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</li> <li><a href="#cite_ref-7">↑</a> 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</li> <li><a href="#cite_ref-8">↑</a> 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</li> <li><a href="#cite_ref-9">↑</a> 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</li> <li><a href="#cite_ref-10">↑</a> 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</li> <li><a href="#cite_ref-11">↑</a> Lee et al. 2008 - Lee J.H., et al, PRX and LXR in hepatic Steatosis: a new dog and an old dog with new tricks, Mol. Pharm., 5(No 1),60-66, 2008</li> <li><a href="#cite_ref-12">↑</a> 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</li> <li><a href="#cite_ref-13">↑</a> 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</li> <li><a href="#cite_ref-14">↑</a> 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</li> <li><a href="#cite_ref-15">↑</a> 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  </li> </ol> NOTE: Italics symbolize edits from John Frisch AOPWiki 2016-11-29T18:41:22

2024-03-26T10:35:31

Increase, Fatty acid influx

Increase, FA influx

Cellular

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 <a href="#cite_note-1">[1]</a>. CD36 has consistently been shown to be expressed at the plasma membrane and to enhance LCFA uptake upon over-expression <a href="#cite_note-2">[2]</a>, <a href="#cite_note-3">[3]</a>.

Increases in fatty acid influx are generally measured by increases in triglycerides, fatty acids, cholesterols, and similar compounds in cells.  In addition, assessment is generally made for plasma membrane stability and/or gene expression increases with genes associated with influx, to associate the increase in fatty acid compounds with influx rather than other pathways (ex. synthesis). Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically to assess levels in control versus potentially affected individuals; common techniques include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, lipidomics, and other direct assessment techniques (Schaefer et al. 2016; Yang and Han 2016).  Analysis is often performed to look at gene expression levels to see which pathway(s) have increased expression levels, to attribute plausibility to changes in influx, eflux, synthesis, and/or breakdown pathways (Nguyen et al. 2008; Mellor et al. 2016, Aguayo-Orozco et al. 2018).  Assessment of cellular components including mitochondria and membrane integrity can also be used as evidence of alteration of normal function within cells.

Life Stage: Older individuals are more likely to manifest this key event  (adults > juveniles) due to increased opportunity to increase fatty acid influx. Sex: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

UBERON:0002107

UBERON liver

CL:0000182

CL hepatocyte

High Unspecific

High

Adult

Moderate

Juvenile

High

<ol> <li><a href="#cite_ref-1">↑</a> 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</li> <li><a href="#cite_ref-2">↑</a> Baranowski 2008 - Baranowski, Biological role of liver X receptors, Journal of Physiology and Pharmacology, 59 (Suppl 7), 31–55, 2008</li> <li><a href="#cite_ref-3">↑</a> 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</li> </ol> Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O.  2018.  Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity.  Frontiers in Genetics 9(Article 396): 1-15. 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. Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H.  2008.  Liver lipid metabolism.  Journal of Animal Physiology and Animal Nutrition 92: 272–283. Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/ Yang, K. and Han, X.  2016.  Lipidomics: Techniques, applications, and outcomes related to biomedical sciences.  Trends in Biochemical Sciences 2016 November ; 41(11): 954–969. NOTE: Italics symbolize edits from John Frisch AOPWiki 2016-11-29T18:41:22

2026-02-11T06:21:24

Activation, ChREBP

Molecular

ChREBP is an LXR target that independently enhances the up-regulation of select lipogenic genes. The up-regulation of the ChREBP target gene is through liver-type pyruvate kinase (L-PK). Therefore, activation of LXR not only increases ChREBP mRNA via enhanced transcription but also modulates its activity <a href="#cite_note-1">[1]</a>. In the liver, ChREBP mediates activation of several regulatory enzymes of glycolysis and lipogenesis including L-PK, acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS). However, according to the study of Denechaud increase in the glucose flux in the cell is a prerequisite for ChREBP activation from T0901317 in mice <a href="#cite_note-2">[2]</a>.

CL:0000182

CL hepatocyte

<ol class="references"> <li id="cite_note-1"><a href="#cite_ref-1">↑</a> Cha & Repa 2007 </li> <li id="cite_note-2"><a href="#cite_ref-2">↑</a> Denechaud et al. 2008 </li> </ol> AOPWiki 2016-11-29T18:41:22

2017-09-16T10:14:53

Activation, SREBP-1c

Molecular

An increase on the mRNA of the SREBP-1c is responsible for an increase of the mRNA of lipogenic enzymes like acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) (Foretz et al. 1999, Foretz et al. 2000). This finding is demonstrated from the absence of triglyceride accumulation on SREBP-1c (-/-) mice <a href="#cite_note-1">[1]</a>, <a href="#cite_note-2">[2]</a>, <a href="#cite_note-3">[3]</a>, <a href="#cite_note-4">[4]</a>. However there is evidence that this effect is not induced in the embryonic state indicating a different role of the SREBP-1c between embryonic and adult life <a href="#cite_note-5">[5]</a>. It is also suggested that for lipogenic genes, SREBP-1c acts together with ChREBP <a href="#cite_note-6">[6]</a>. In addition, in STZ diabetic mice, adenovirus-mediated over-expression of SREBP-1c in the liver resulted in an increase of lipogenic enzyme expression with an increase of the triglyceride hepatic content and a marked decrease in the hyperglycaemia of diabetic mice mimicking perfectly the effect of an insulin injection <a href="#cite_note-7">[7]</a>. Finally there are a number of studies that demonstrated that SREBP-1c is essential for glucokinase (GK) expression and that it is a mediator of insulin action <a href="#cite_note-8">[8]</a>, <a href="#cite_note-9">[9]</a>.

CL:0000182

CL hepatocyte

<ol class="references"> <li id="cite_note-1"><a href="#cite_ref-1">↑</a> Liang et al. 2002 </li> <li id="cite_note-2"><a href="#cite_ref-2">↑</a> Schultz et al. 2000 </li> <li id="cite_note-3"><a href="#cite_ref-3">↑</a> Horton et al. 2002 </li> <li id="cite_note-4"><a href="#cite_ref-4">↑</a> Shimano et al. 1999 </li> <li id="cite_note-5"><a href="#cite_ref-5">↑</a> Liang et al. 2002 </li> <li id="cite_note-6"><a href="#cite_ref-6">↑</a> Ishii et al. 2004 </li> <li id="cite_note-7"><a href="#cite_ref-7">↑</a> Bécard et al. 2001 </li> <li id="cite_note-8"><a href="#cite_ref-8">↑</a> Ferre 2007 </li> <li id="cite_note-9"><a href="#cite_ref-9">↑</a> Fleischmann 1999 </li> </ol> AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:53

Activation, FAS

Molecular

LXR agonist treatment has been shown to induce the genes encoding fatty acid synthase (FAS) in SREBP-1c-deficient mice <a href="#cite_note-1">[1]</a>, <a href="#cite_note-Liang_et_al._2002-2">[2]</a>, <a href="#cite_note-Schultz_et_al._2000-3">[3]</a>. This finding shows that in parallel with the increase of FAS expression from the SREBP-1c <a href="#cite_note-Liang_et_al._2002-2">[2]</a>, <a href="#cite_note-Schultz_et_al._2000-3">[3]</a>, <a href="#cite_note-4">[4]</a> and the ChREBP the enzyme is also directly induced from the LXR.

CL:0000182

CL hepatocyte

<ol class="references"> <li id="cite_note-1"><a href="#cite_ref-1">↑</a> Oisterveer et al. 2010 </li> <li id="cite_note-Liang_et_al._2002-2">↑ <a href="#cite_ref-Liang_et_al._2002_2-0">2.0</a> <a href="#cite_ref-Liang_et_al._2002_2-1">2.1</a> Liang et al. 2002 </li> <li id="cite_note-Schultz_et_al._2000-3">↑ <a href="#cite_ref-Schultz_et_al._2000_3-0">3.0</a> <a href="#cite_ref-Schultz_et_al._2000_3-1">3.1</a> Schultz et al. 2000 </li> <li id="cite_note-4"><a href="#cite_ref-4">↑</a> Horton et al. 2002 </li> </ol> AOPWiki 2016-11-29T18:41:22

2017-09-16T10:14:54

Activation, SCD-1

Molecular

In addition to the FAS gene induction LXR activation leads to the direct induction of the stearoyl-CoA desaturase 1 (SCD1) in SREBP-1c-deficient mice <a href="#cite_note-1">[1]</a>, <a href="#cite_note-2">[2]</a>, <a href="#cite_note-3">[3]</a>. The role of SCD-1 could be crucial for the lipogenic activity of LXRs as there are data supporting that SCD-1 deficient mice are completely protected against hypertriglyceridemia and TG accumulation in liver is decreased after treatment with T0901317 <a href="#cite_note-4">[4]</a>.

CL:0000182

CL hepatocyte

<ol class="references"> <li id="cite_note-1"><a href="#cite_ref-1">↑</a> Oisterveer et al. 2010 </li> <li id="cite_note-2"><a href="#cite_ref-2">↑</a> Liang et al. 2002 </li> <li id="cite_note-3"><a href="#cite_ref-3">↑</a> Schultz et al. 2000 </li> <li id="cite_note-4"><a href="#cite_ref-4">↑</a> Chu et al. 2006 </li> </ol> AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:54

Synthesis, De Novo Fatty Acid (FA)

Cellular

A number of pathways and a great number of enzymes like GK, L-PK, ACC, FAS and SCD-1 are involved in the de novo FA synthesis <a href="#cite_note-Postic_.26_Girard_2008-1">[1]</a>. As it is already discussed above these enzymes are induced by LXR agonists (FAS, SCD1), the SREBP-1c (GK, ACC, FAS) and the ChREBP (L-PK, ACC, FAS) leading to enhancement of the de novo FA synthesis. <a class="image" href="/wiki/index.php/File:Metabolic-pathway-for-de-novo-FA-synthesis-and-TG-formation.png"><img alt="Metabolic-pathway-for-de-novo-FA-synthesis-and-TG-formation.png" src="/wiki/images/5/5c/Metabolic-pathway-for-de-novo-FA-synthesis-and-TG-formation.png" style="height:556px; width:879px" /></a> Figure 1. Metabolic pathway for de novo FA synthesis and TG formation <a href="#cite_note-Postic_.26_Girard_2008-1">[1]</a> As proposed from Diraison et al 1997 the de novo FA synthesis contributes maximum 5% to the synthesis of FA and TG under normal conditions. Conditions associated with high rates of lipogenesis, such as low fat - high carbohydrate (LF/HC) diet, hyperglycemia, and hyperinsulinemia are associated with a shift in cellular metabolism from lipid oxidation to TG esterification, thereby increasing the availability of TGs derived from VLDL synthesis and secretion.

Increases in fatty acid synthesis are generally measured by increases in triglycerides, fatty acids, cholesterols, and similar compounds in cells.  In addition, assessment is generally made for cellular components such as mitochondria and/or gene expression increases with genes associated with synthesis, to associate the increase in fatty acid compounds with synthesis rather than other pathways (ex. influx). Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically to assess levels in control versus potentially affected individuals; common techniques include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, lipidomics, and other direct assessment techniques (Schaefer et al. 2016; Yang and Han 2016).  Analysis is often performed to look at gene expression levels to see which pathway(s) have increased expression levels, to attribute plausibility to changes in influx, eflux, synthesis, and/or breakdown pathways (Nguyen et al. 2008; Mellor et al. 2016, Aguayo-Orozco et al. 2018).  Assessment of cellular components including mitochondria and membrane integrity can also be used as evidence of alteration of normal function within cells.

Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides. Sex: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

CL:0000182

CL hepatocyte

High Unspecific

High

Adult

Moderate

Juvenile

High

<ol> <li>↑ <a href="#cite_ref-Postic_.26_Girard_2008_1-0">1.0</a> <a href="#cite_ref-Postic_.26_Girard_2008_1-1">1.1</a> Postic & Girard 2008 - Postic C., Girard J., Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice, J. Clin. Invest. 118 (No 3), 829–838, 2008</li> <li>Diraison et al 1997 - Diraison F., et al, Role of human liver lipogenesis and re-esterification in triglycerides secretion and in FFA re-esterification. Am J Physiol., 274 (2 Pt 1), E321-327, 1998</li> </ol> Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O.  2018.  Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity.  Frontiers in Genetics 9(Article 396): 1-15. 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. Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H.  2008.  Liver lipid metabolism.  Journal of Animal Physiology and Animal Nutrition 92: 272–283. Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/ Yang, K. and Han, X.  2016.  Lipidomics: Techniques, applications, and outcomes related to biomedical sciences.  Trends in Biochemical Sciences 2016 November ; 41(11): 954–969. NOTE: Italics symbolize edits from John Frisch AOPWiki 2016-11-29T18:41:22

2024-03-29T10:55:15

Accumulation, Triglyceride

Cellular

Triglycerides are important building blocks for a wide variety of compounds found in organisms, with cellular concentrations reflecting the relative rate of influx and efflux, as well as the relative rate of synthesis and breakdown.  However, excess accumulation leads to Fatty Liver Cells and steatosis. In this key event we focus on excessive accumulation of triglycerides in mammalian systems.  Four major pathways for triglyceride accumulation are: 1. Increased fatty acid uptake; 2. Increased De Novo FA and Lipid Synthesis; 3. Decreased FA Oxidation; 4. Decreased Lipid Efflux (Angrish et al. 2016).  Chemical stressors can increase gene expression of key genes involving these pathways, leading to increased accumulation of triglycerides (Aguayo-Orozco et al. 2018).  In addition, excessive dietary compounds of fatty compounds can also increase likelihood of accumulation of triglycerides (Nguyen et al. 2008).  Nuclear receptors that have been implicated in causing excessive accumulation of triglycerides leading to steatosis, when overexpressed, include (Mellor et al. 2016): Aryl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GXR), Liver X receptor (LXR), Peroxisome proliferator-activated receptor (PPAR), Pregnane X receptor (PXR), and Retinoic acid receptor (RAR or RXR).

Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically to assess levels in control versus potentially affected individuals; common techniques include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, lipidomics, and other direct assessment techniques (Schaefer et al. 2016; Yang and Han 2016).  Analysis is often performed to look at gene expression levels to see which pathway(s) have increased expression levels, to attribute plausibility to changes in influx, eflux, synthesis, and/or breakdown pathways (Nguyen et al. 2008; Mellor et al. 2016, Aguayo-Orozco et al. 2018).  Assessment of cellular components including mitochondria and membrane integrity can also be used as evidence of alteration of normal function within cells.

Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to accumulation of triglycerides. Sex: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).  Likely pervasive in many animal taxa.

CL:0000182

CL hepatocyte

High Unspecific

High

Adult

Moderate

Juvenile

High

Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O.  2018.  Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity.  Frontiers in Genetics 9(Article 396): 1-15. 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. 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. Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H.  2008.  Liver lipid metabolism.  Journal of Animal Physiology and Animal Nutrition 92: 272–283. Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/ Yang, K. and Han, X.  2016.  Lipidomics: Techniques, applications, and outcomes related to biomedical sciences.  Trends in Biochemical Sciences 2016 November ; 41(11): 954–969. NOTE: Italics symbolize edits from John Frisch AOPWiki 2016-11-29T18:41:23

2024-03-26T13:09:28

N/A, Liver Steatosis

Organ

UBERON:0002107

UBERON liver

AOPWiki 2016-11-29T18:41:24

2017-09-16T10:14:55

peroxisome proliferator activated receptor promoter demethylation

demethylation, PPARg promoter

Molecular

Biological state The Peroxisome Proliferator Activated receptor γ (PPARγ) belongs to <a href="/wiki/index.php/Peroxisome_Proliferator_Activated_receptors_(PPARs;_NR1C)" title="Peroxisome Proliferator Activated receptors (PPARs; NR1C)">Peroxisome Proliferator Activated receptors (PPARs; NR1C)</a> steroid/thyroid/retinoid receptor superfamily of transcription factors, which respond to specific ligands by altering gene expression in a cell-specific manner. The PPARγ gene contains three promoters that yield three isoforms, namely, PPAR-γ1, 2 and 3. PPAR-γ1 and γ3 RNA transcripts translate into the identical PPAR-γ1 protein. Biological compartments PPARγ is abundantly expressed in adipose tissue, promoting adipocyte differentiation, but is also present in various cells and tissues, for review see (Braissant et al. 1996). PPARγ expression is tissue dependent (L Fajas et al. 1997), (Lluis Fajas, Fruchart, and Auwerx 1998). PPARγ is most highly expressed in white adipose tissue and brown adipose tissue, where it is a master regulator of adipogenesis as well as a potent modulator of whole-body lipid metabolism and insulin sensitivity (Evans, Barish, and Wang 2004), (Tontonoz and Spiegelman 2008). Whereas PPARγ1 is expressed in many tissues, the expression of PPARγ2 is restricted to adipose tissue under physiological conditions but can be induced in other tissues by a high-fat diet (Saraf et al. 2012). General role in biology PPARγ is activated after the binding of natural ligands such as polyunsaturated fatty acids and prostaglandin metabolites. It can also be activated by synthetic ligands such as thiazolidinediones (TZDs) (rosiglitazone, pioglitazone or troglitazone) (Lehmann et al., 1995). PPARγ controls many vital processes such as glucose metabolism and inflammation as well as variety of developmental programs(Wahli & Desvergne, 1999), (Rotman et al., 2008), (Wahli & Michalik, 2012). This receptor itself is essential for developmental processes since targeted disruption of this gene results in embryo lethality, due in part to defective placental development, therefore modulation of PPARγ activity may impact endocrine regulated processes during development as well as later in life.

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? Binding of ligands to PPARγ is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from the transactivation using e.g. reporter assay with a reporter gene that demonstrates functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change promoting binding to transcriptional coactivators. Conversely, binding of antagonists results in a conformation that favours the binding of corepressors (Yu & Reddy, 2007) (Viswakarma et al., 2010. Transactivation assays are performed using the transient or stably transfected cells with the PPARγ expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPARγ activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARγ transcription factor assay kits, see Table 1. The transactivation (stable transfection) assay provides the most applicable OECD Level 2 assay aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, & Kloas, 2011). Currently no internationally validated assays are available.     <table class="wikitable" id="Event228"> <tbody> <tr> <th>Key event</th> <th colspan="7">PPARγ activation</th> </tr> <tr> <td> </td> <td>What is measured?</td> <td>Ligand Binding</td> <td> </td> <td>Transcriptional activity</td> <td> </td> <td> </td> <td> </td> <td> </td> </tr> <tr> <td>Method/test category</td> <td>molecular modelling</td> <td>binding assay</td> <td>transactivation reporter gene assay</td> <td> </td> <td> </td> <td>transcription factor assay</td> <td> </td> </tr> <tr> <td>Method/test name</td> <td>molecular modelling; docking</td> <td>Scintillation proximity binding assay</td> <td>luciferase reporter gene assay</td> <td> </td> <td> </td> <td>PPARγ (mouse/rat) Reporter Assay Kit</td> <td>Electrophoretic Mobility Shift Assay (EMSA)</td> </tr> <tr> <td>Test environment</td> <td>In silico</td> <td>In vitro</td> <td>In vitro</td> <td> </td> <td> </td> <td>In vitro, ex vivo</td> <td> </td> </tr> <tr> <td>Test principle</td> <td>Computational simulation of a candidate ligand binding to a receptor, Predicts the strength of association or binding affinity.</td> <td>direct binding indicating the mode of action for PPARα/γ</td> <td>Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity.</td> <td> </td> <td> </td> <td>PPARγ once activated by a ligand, the receptor binds to a promoter element in the gene for target gene and activates its transcription. The bound (activated) to DNA PPAR is measured.</td> <td> </td> </tr> <tr> <td>Test outcome</td> <td>A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. If the protein is a receptor, ligand binding may result in agonism or antagonism</td> <td>Assess the ability of compounds to bind to PPARγ. Identifies the modulators of PPARγ.</td> <td>The changes in activity of reporter gene levels functionally linked to a PPAR-responsive element/promoter gives information about the activity of the PPAR activation.</td> <td> </td> <td> </td> <td>Protein: DNA binding, DNA binding activity</td> <td> </td> </tr> <tr> <td>Test background</td> <td>Predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using, for example, scoring functions.</td> <td>This assay determines whether compounds interact directly with PPARγ.</td> <td>PPARγ COS-1cell transactivation assay (transient transfection with human or mouse PPARγ expression plasmid and pHD(x3)-Luc reporter plasmid</td> <td>(PPRE)3- luciferase reporter construct C2C12</td> <td>Proprietary rodent cell line expressing the mouse/rat PPARγ</td> <td>Transcriptional activity of PPARγ can be assessed using commercially available kits like e.g. PPARγ transcription factor assay kit (Abcam, Cambridge, USA or Cayman Chemical, USA).</td> <td>Gene regulation and determining protein: DNA interactions are the detected by the EMSA. EMSA can be used qualitatively to identify sequence-specific DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences within a given genes upstream regulatory region. EMSA can also be utilized quantitatively to measure thermodynamic and kinetic parameters.</td> </tr> <tr> <td>Assay type</td> <td>Quantitative</td> <td>Qualitative</td> <td>Quantitative</td> <td>Quantitative</td> <td>Quantitative</td> <td>Quantitative</td> <td>Quantitative</td> </tr> <tr> <td>Application domain</td> <td>Virtual screening</td> <td>In vitro screening</td> <td>In vitro Screening, functional studies activity (reported use: agonist)</td> <td> </td> <td>In vitro Screening functional activity (antagonist/agonist)</td> <td>Functional studies</td> <td>Functional studies</td> </tr> <tr> <td>Source</td> <td>Research/commercial</td> <td>Research</td> <td>Research</td> <td>Research</td> <td>commercial</td> <td>commercial</td> <td>Research/commercial</td> </tr> <tr> <td>Ref</td> <td>(Feige et al., 2007), (Kaya, Mohr, Waxman, & Vajda, 2006)</td> <td>(Lapinskas et al., 2005), (Wu, Gao, & Wang, 2005)</td> <td>(Maloney & Waxman, 1999)</td> <td>(Feige et al., 2007)</td> <td>Cayman, (Gijsbers et al. 2013)</td> <td>Abcam<a class="external autonumber" href="http://www.abcam.com/ppar-gamma-transcription-factor-assay-kit-ab133101.html" rel="nofollow" target="_blank">[1]</a></td> <td> </td> </tr> </tbody> </table> Table 1 Summary of the chosen methods to measure the PPARγ activation.

PPARγ have been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (Wahli & Desvergne, 1999).

CL:0000182

CL hepatocyte

High Moderate Moderate

Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., … Evans, R. M. (1999). PPAR gamma is required for placental, cardiac, and adipose tissue development. Molecular Cell, 4(4), 585–95. Braissant, O., Foufelle, F., Scotto, C., Dauça, M., & Wahli, W. (1996). Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology, 137(1), 354–66. Burns, K. A., & Vanden Heuvel, J. P. (2007). Modulation of PPAR activity via phosphorylation. Biochimica et Biophysica Acta, 1771(8), 952–60. doi:10.1016/j.bbalip.2007.04.018 Fajas, L., Auboeuf, D., Raspé, E., Schoonjans, K., Lefebvre, A. M., Saladin, R., … Auwerx, J. (1997). The organization, promoter analysis, and expression of the human PPARgamma gene. The Journal of Biological Chemistry, 272(30), 18779–89. Fajas, L., Fruchart, J.-C., & Auwerx, J. (1998). PPARγ3 mRNA: a distinct PPARγ mRNA subtype transcribed from an independent promoter. FEBS Letters, 438(1-2), 55–60. doi:10.1016/S0014-5793(98)01273-3 Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions. Progress in Lipid Research, 45(2), 120–59. doi:10.1016/j.plipres.2005.12.002 Feige, J. N., Gelman, L., Rossi, D., Zoete, V., Métivier, R., Tudor, C., … Desvergne, B. (2007). The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis. The Journal of Biological Chemistry, 282(26), 19152–66. doi:10.1074/jbc.M702724200 Gijsbers, Linda, Henriëtte D L M van Eekelen, Laura H J de Haan, Jorik M Swier, Nienke L Heijink, Samantha K Kloet, Hai-Yen Man, et al. 2013. “Induction of Peroxisome Proliferator-Activated Receptor Γ (PPARγ)-Mediated Gene Expression by Tomato (Solanum Lycopersicum L.) Extracts.” Journal of Agricultural and Food Chemistry 61 (14) (April 10): 3419–27. doi:10.1021/jf304790a. Issemann, I., & Green, S. (1990). Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature, 347(6294), 645–650. Kaya, T., Mohr, S. C., Waxman, D. J., & Vajda, S. (2006). Computational screening of phthalate monoesters for binding to PPARgamma. Chemical Research in Toxicology, 19(8), 999–1009. doi:10.1021/tx050301s Lapinskas, P. J., Brown, S., Leesnitzer, L. M., Blanchard, S., Swanson, C., Cattley, R. C., & Corton, J. C. (2005). Role of PPARα in mediating the effects of phthalates and metabolites in the liver. Toxicology, 207(1), 149–163. Le Maire, A., Grimaldi, M., Roecklin, D., Dagnino, S., Vivat-Hannah, V., Balaguer, P., & Bourguet, W. (2009). Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Reports, 10(4), 367–73. doi:10.1038/embor.2009.8 LeBlanc, G., Norris, D., & Kloas, W. (2011). Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors, (178). Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., & Kliewer, S. A. (1995). An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor (PPAR ). Journal of Biological Chemistry, 270(22), 12953–12956. doi:10.1074/jbc.270.22.12953 Maloney, E. K., & Waxman, D. J. (1999). trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals. Toxicology and Applied Pharmacology, 161(2), 209–218. Michalik, L., Zoete, V., Krey, G., Grosdidier, A., Gelman, L., Chodanowski, P., … Michielin, O. (2007). Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior. The Journal of Biological Chemistry, 282(13), 9666–77. doi:10.1074/jbc.M610523200 Morán-Salvador, E., López-Parra, M., García-Alonso, V., Titos, E., Martínez-Clemente, M., González-Périz, A., … Clària, J. (2011). Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 25(8), 2538–50. doi:10.1096/fj.10-173716 Pereira-Fernandes, A., Demaegdt, H., Vandermeiren, K., Hectors, T. L. M., Jorens, P. G., Blust, R., & Vanparys, C. (2013). Evaluation of a screening system for obesogenic compounds: screening of endocrine disrupting compounds and evaluation of the PPAR dependency of the effect. PloS One, 8(10), e77481. doi:10.1371/journal.pone.0077481 ToxCastTM Data, US Environmental Protection Agency. <a class="external free" href="http://www.epa.gov/ncct/toxcast/data.html" rel="nofollow" target="_blank">http://www.epa.gov/ncct/toxcast/data.html</a>. Vanden Heuvel, J. P. (1999). Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis. Toxicological Sciences : An Official Journal of the Society of Toxicology, 47(1), 1–8. Viswakarma, N., Jia, Y., Bai, L., Vluggens, A., Borensztajn, J., Xu, J., & Reddy, J. K. (2010). Coactivators in PPAR-Regulated Gene Expression. PPAR Research, 2010. doi:10.1155/2010/250126 Wahli, W., & Desvergne, B. (1999). Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews, 20(5), 649–88. Wu, B., Gao, J., & Wang, M. (2005). Development of a complex scintillation proximity assay for high-throughput screening of PPARgamma modulators. Acta Pharmacologica Sinica, 26(3), 339–44. doi:10.1111/j.1745-7254.2005.00040.x Yu, S., & Reddy, J. K. (2007). Transcription coactivators for peroxisome proliferator-activated receptors. Biochimica et Biophysica Acta, 1771(8), 936–51. doi:10.1016/j.bbalip.2007.01.008 AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:53

Increase, Mitochondrial dysfunction

Cellular

Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress. Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006). Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation. A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010). Metal-induced Mitochondrial Dysfunction Mitochondria are an important site of Ca2+ regulation and storage, taking up Ca2+ ions electrophoretically from the cytosol through a Ca2+ uniporter, which can then accumulate in the mitochondria (Roos et al., 2012; Orrenius et al., 2015). Similarities between calcium and metals, such as cadmium and lead, makes the entrance and accumulation of these metals into the mitochondria via calcium metals possible by mode of molecular mimicry (Mathews et al., 2013; Adiele et al., 2012). The outer mitochondrial membrane also contains the divalent metal transporter (DMT1), which allows for mitochondrial uptake of divalent metals such as Fe and Mn. When cells are under heavy metal-induced stress, DMT has been shown to be overexpressed in the mitochondrial membrane, making the mitochondria targets of metal toxicity and accumulation. Heavy metal exposure in aerobic organisms increases ROS formation through redox cycling, where metals with different valence states (Fe, Cu, Cr, etc.) directly produce ROS as they are reduced by cellular antioxidants and then react with oxygen (Shaki et al., 2012; Shaki et al., 2013; Pourahmad et al., 2006; Santos et al., 2007). The production of highly reactive hydroxyl radicals under mitochondrial oxidative stress and in the presence of transition metals occurs via the Fenton reaction or Haber-Weiss reaction (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Metals and ROS are capable of damaging mitochondrial DNA as well as mechanisms of DNA repair and proliferation arrest (Valko et al., 2005). Metals and ROS have the potential to directly damage mitochondrial membranes and structure by binding to and oxidizing membrane lipids and proteins. This structural damage can collapse the MMP and lead to the opening of the MPTP (Orrenius et al., 2015; Roos et al., 2012; Pourahmad et al., 2006). Uranium and mercury, for example, have both been shown to directly inhibit the mitochondrial electron transport chain and interfere with ATP production (Shaki et al., 2012; Roos et al., 2012). Furthermore, as previously mentioned, metals have been shown to inhibit ROS-detoxifying enzymes. By binding to these enzymes, metals can inhibit their antioxidant functions, and cause an accumulation of ROS and increased synthesis of more antioxidant enzymes in order to combat the oxidative stress (Blajszczak and Bonini, 2017). Summing up: Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.

Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail. I. Mitochondrial dysfunction assays assessing a loss-of function. 1. Cellular oxygen consumption. See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O2 consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005). 2. Mitochondrial membrane potential (Δψm ). - Revision of AOP3 (Project: <a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms" rel="noreferrer noopener" target="_blank">NP/EFSA/PREV/2024/02</a>): The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. Quantitative assessment of ΔΨm in living cells is most commonly achieved through the use of cationic, lipophilic fluorescent probes that accumulate within the mitochondrial matrix in proportion to the electrochemical gradient (Leonard et al., 2014). Among these, tetramethylrhodamine derivatives such as TMRE (tetramethylrhodamine ethyl ester) and TMRM (tetramethylrhodamine methyl ester) are widely employed due to their reversible, potential-dependent distribution across the inner mitochondrial membrane (Scaduto and Grotyohann, 1999; Creed and McKenzie, 2019). When applied at non-quenching, nanomolar concentrations, these dyes allow linear and quantitative detection of ΔΨm, as fluorescence intensity directly correlates with mitochondrial polarization. Detection can be performed by flow cytometry for population-level quantification, by high-content microscopy for spatially resolved analysis, or by fluorescence plate readers for higher throughput (Wong and Cortopassi, 2002; Valdebenito and Dunchen, 2022). Quantitative interpretation requires the use of appropriate controls, typically involving treatment with protonophores such as FCCP or CCCP, which fully dissipate ΔΨm and thereby establish baseline fluorescence, and inhibitors such as oligomycin or antimycin A to reveal different components of mitochondrial respiration. In parallel, dyes such as JC-1 are also used, though their ratiometric readout is less sensitive at low potentials and more prone to artifacts compared with TMRE or TMRM (Leonard et al., 2022). For accurate normalization, measurements are often corrected for cell number, mitochondrial content, or total protein, and fluorescence changes are expressed relative to maximal depolarization. In addition to chemical probes, genetically encoded sensors, such as mitochondria-targeted fluorescent proteins fused to potential-sensitive domains, provide complementary tools for ΔΨm monitoring in live-cell and in vivo contexts (Leonard et al., 2022). - Not endorsed  3. Enzymatic activity of the electron transport system (ETS). Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990). 4. ATP content. For the evaluation of ATP levels, various commercially-available ATP assay kits are offered  based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005). <div> - Revision of AOP3 (Project: <a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms">NP/EFSA/PREV/2024/02</a>): Determination of mitochondrial ATP production based on extracellular flux analysis   The method is based on the detection of OCR (Oxygen Consumption Rate) that represents mitochondrial respiration as well as on the detection of ECAR (extracellular acidification rate) / proton efflux rate (PER): reflects extracellular acidification, a proxy for glycolysis (lactate release) plus contributions from CO₂/HCO₃⁻. PER is preferred over raw ECAR since it corrects for CO₂-derived acidification (Desousa et al., 2023; Espinosa et al., 2022). Application of inhibitors of individual complexes of the respiratory chain allows the detection of ATP-linked OCR: portion of oxygen consumption directly driving ATP synthesis (lost after ATP synthase inhibition) (Yoo et al., 2024). The proton leak & non-mitochondrial OCR represents remaining oxygen consumption after ATP synthase and electron transport chain inhibitor addition. The difference yields the ATP-coupled respiration component.   Calculation of mitochondrial ATP production  Mito ATP production rate (pmol ATP/min) = OCRATP (pmol O2/min) × 2 × P/O    OCR_ATP: ATP-coupled portion of OCR.   Factor 2: each O₂ molecule contains two oxygen atoms.   P/O ratio: number of ATP molecules synthesized per oxygen atom reduced. A mean P/O ≈ 2.75 is typically assumed (validated across many cell types but substrate- and condition-dependent) (Plitzko and Loesgen, 2018; Mookerjee et al., 2017; Motawe et al., 2024).    Limitations   P/O ratio varies by substrate (glucose vs. fatty acids), cell type, and conditions. Fixed values are approximations.   Non-mitochondrial oxygen consumption (oxidases, peroxidases, etc.) can confound OCR, hence use of ETC inhibitors.   PER vs. ECAR: CO₂-driven acidification must be corrected to avoid overestimating glycolytic ATP.   Normalization: results are usually expressed per cell, protein content, DNA, or mitochondrial mass — interpretation depends on normalization method.  - Not endorsed </div> II. Mitochondrial dysfunction assays assessing a gain-of function. 1. Mitochondrial permeability transition pore opening (PTP). The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999). 2. mtDNA damage as a biomarker of mitochondrial dysfunction. Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b). 3. Generation of ROS and resultant oxidative stress. a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine. b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential). GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress. c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method. d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (<a class="external free" href="http://www.biotek.com/resources/articles/reactive-oxygen-species.html" rel="nofollow" target="_blank">http://www.biotek.com/resources/articles/reactive-oxygen-species.html</a>). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria. e. Detection of hydrogen peroxide (H2O2) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product (Tarpley et al., 2004). Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (mPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion) (Promega assay, TB369; Radkowsky et al., 1986) • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent. All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE <a href="/wiki/index.php/Event:209" title="Event:209"> Oxidative Stress, Increase</a> <table border="1" cellpadding="1" cellspacing="1"> <tbody> <tr> <td> Assay Type & Measured Content </td> <td>Description</td> <td>Dose Range Studied</td> <td> Assay Characteristics (Length/Ease of use/Accuracy) </td> </tr> <tr> <td> Rhodamine 123 Assay Measuring Mitochondrial membrane potential (MMP) and its collapse  (Shaki et al., 2012) </td> <td> Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively. </td> <td>50, 100 and 500 μM of uranyl acetate</td> <td> Short / easy Medium accurancy </td> </tr> <tr> <td> TMRE fluorescence Assay Measuring Mitochondrial permeability transition pore (mPTP) opening (Huser et al., 1998) </td> <td>Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore.</td> <td>1 µM cyclosporin A</td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> GSH / GSSG Determination Assay Measuring  cellular glutathione (GSH) status; ratio of GSH/GSSG (Owen & Butterfield, 2010; Shaki et al., 2013) </td> <td>GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer.</td> <td>100 µM uranyl acetate</td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> TBARS Assay Quantification of lipid peroxidation (Yuan et al., 2016) </td> <td>MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm.</td> <td>200, 400, 800 µM uranyl acetate</td> <td> Medium / medium High accurancy </td> </tr> <tr> <td> Aequorin-based bioluminescence assay Increase in mitochondrial Ca2+ influx (Pozzan & Rudolf, 2009) </td> <td>Together with GFP, the aequorin moiety acts as Ca2+ sensor in vivo, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.</td> <td> </td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> Western blot & immunostaining analyses Measuring cytochrome c release (Chen et al., 2000)</td> <td>Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)</td> <td> </td> <td> Short / easy Medium accurancy </td> </tr> <tr> <td> Quantikine Rat/Mouse Cytochrome c Immunoassay Measuring cytochrome c release (Shaki et al., 2012) </td> <td>Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.</td> <td> </td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> Membrane potential and cell viability – Flow Cytometry Measuring cytochrome c release (Kruidering et al., 1997) </td> <td>“Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 g. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of 60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water et al., 1993)”</td> <td> </td> <td> Short / easy Medium accurancy </td> </tr> </tbody> </table>

Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010). - Revision of AOP3 (Project: <a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms" rel="noreferrer noopener" target="_blank">NP/EFSA/PREV/2024/02</a>): Endogenous ROS formation by complex I: In mammals, complex I is a dominant site of mitochondrial ROS, especially via RET. In plants (Senkler et al. 2017; Maldonado), mitochondria contain alternative NAD(P)H dehydrogenases and an alternative oxidase (AOX) that bypass Complex I and III These pathways reduce ROS formation by preventing over-reduction of the ETC. Complex I still produces ROS, but generally less damaging due to AOX. Yeast: S. cerevisiae lacks a canonical Complex I entirely, relying instead on alternative NADH dehydrogenases. Consequently, mitochondrial ROS production from a Complex I-like source is absent. Other fungi with true Complex I (e.g., Neurospora crassa) do generate ROS similar to animals. - Not endorsed

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2026-02-11T07:06:25

<upstream-id>819f1636-046f-4794-b303-8f5d104cd22c</upstream-id> <downstream-id>552697e1-19cf-4b16-9139-6e0c62c70a1a</downstream-id>

AOPWiki 2016-11-29T18:41:33

2016-12-03T16:37:55

<upstream-id>552697e1-19cf-4b16-9139-6e0c62c70a1a</upstream-id> <downstream-id>4d6be341-6962-45a3-b40e-ef76752deace</downstream-id> CD36 gene expression has been shown to be a key regulator of fatty acid influx, primarily in mammal studies.  CD36 is a transmembrane protein, and increased CD36 gene expression can result in increased fatty acid influx.  Chemical stressors or high fat diets can help trigger fatty acid influx.

This KER was identified as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki. Support for this KER is referenced in publications cited in the originating work of Landesmann et al. (2012) and Negi et al. (2021).

The biological plausibility linking increased CD36 expression to increased fatty acid uptake is moderate.  CD36 is a transmembrane protein, and upregulation of CD36 has been linked to increased fatty acid uptake, primarily in mammalian systems.

Since the link between upregulation of CD36 and increased fatty acid influx has been established, empirical studies often measure increased CD36 gene expression and increased lipid content in cells and infer that the mechanism was increased fatty acid influx (Moya et al. 2010).   <table cellspacing="0" class="Table" style="border-collapse:collapse"> <tbody> <tr> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:97px"> Species </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:69px"> Duration </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:110px"> Dose </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:89px"> Upregulated CD36? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:71px"> Increase FA influx? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:109px"> Summary </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:77px"> Citation </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> Lab mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 5 weeks </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:110px"> Wild-type versus transgenic-human PXR mice. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:89px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:71px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> Transgenic-human PXR mice showed increased expression of CD36 genes in livers and increased lipid accumulation versus wild-type mice.  FA influx was inferred as there was no increase in gene expression of SREBP, which would be expected to be upregulated if de novo fatty acid synthesis was the mechanism for increased triglycerides. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:77px"> Zhou et al. (2006) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> Human (Homo sapiens) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:110px"> Children and adolescents  exhibiting steatosis versus children and adolescents without steatosis </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:89px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:71px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> CD36, FABPpm, SLC27A2, SLC27A5 gene expression were upregulated and CD36 and CPT-1 protein expression was upregulated in subjects exhibiting steatosis linking increased triglyceride levels to fatty acid influx; FASN, SCD1, and acyl-COA gene expression were also upregulated in subjects exhibiting steatosis linking increased triglyceride levels to de novo fatty acid synthesis; both pathways appear to be responsible for increased triglycerides. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:77px"> Zhu et al. (2011) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> Mouse (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 5 weeks </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:110px"> High fat versus low fat diet, transgenic mice </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:89px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:71px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> Mouse fed high fat diet had higher CD36 expression and triglyceride accumulation than mice fed low fat diet; transgenic mice and hepatocytes with CD36 gene had higher fatty acid influx than null mice and  hepatocytes measured by the fluorescent fatty acid analog BODIPY. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:77px"> Koonen et al. (2007) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> Lab mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 24 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:110px"> 20 um efavirenz in vitro </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:89px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:71px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> Increased CD36 gene expression vs control in hepatocytes exposed to 20 um efavirenz and correlated higher fatty acid transport as measured by palmitic acid uptake. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:77px"> Gwag et al. (2009) </td> </tr> </tbody> </table>

Moderate Unspecific

High

Adult

Moderate

Juvenile

Moderate Moderate

Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides. Sex: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C.  2019.  Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis Journal of Hepatology 70: 930–940. Koonen, D.P.Y., Jacobs, R.L., Febbraio, M. Young, M.E., Soltys, C.-L.M., Ong, H., Vance, D.E., and Dyck, J.R.B.  2007.  Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity.  Diabetes 56: 2863-2871. Landesmann, B., Goumenou, M., Munn, S., and Whelan, M.  2012.  Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity.  European Commission Report EUR 25631, 49 pages.  https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en Moya, M., Gomez-Lechon, M.J., Castell, J.V., and Jover, R.  2010.  Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile.  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. Zhu, L., Baker, S.S., Liu, W., Tao, M.-H., Patel, R., Nowak, N.J., and Baker, R.D.  2011.  Lipid in the livers of adolescents with nonalcoholic steatohepatitis: combined effects of pathways on steatosis.  Metabolism Clinical and Experimental 60: 1001-1011. 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.  The Journal of Biological Chemistry 281(21): 15013–15020. AOPWiki 2016-11-29T18:41:33

2024-03-27T10:00:54

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AOPWiki 2016-11-29T18:41:33

2016-12-03T16:37:55

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AOPWiki 2016-11-29T18:41:33

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AOPWiki 2016-11-29T18:41:33

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AOPWiki 2016-11-29T18:41:33

2016-12-03T16:37:54

<upstream-id>4860bd17-7f7f-433a-88db-7f43093d4ed3</upstream-id> <downstream-id>d0ae875b-d65a-4557-9e46-e8c5761d63f3</downstream-id>

AOPWiki 2016-11-29T18:41:33

2016-12-03T16:37:56

<upstream-id>ba7c6967-d68b-4d6a-a486-e215fea7d5f9</upstream-id> <downstream-id>d0ae875b-d65a-4557-9e46-e8c5761d63f3</downstream-id>

AOPWiki 2016-11-29T18:41:33

2016-12-03T16:37:55

<upstream-id>d0ae875b-d65a-4557-9e46-e8c5761d63f3</upstream-id> <downstream-id>7841b130-5332-458d-a955-36a3916798ca</downstream-id> De novo fatty acid synthesis is a main pathway broadly accepted as a mechanism for accumulation of triglycerides in cells.  Chemical stressors or alteration of gene expression levels can trigger increased fatty acid influx, as well as changes to membrane permeability and membrane proteins that facilitate fatty acid transport.

This KER was identified as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki. Support for this KER is referenced in publications cited in the originating work of Landesmann et al. (2012) and Negi et al. (2021).

The biological plausibility linking increased fatty acid synthesis to accumulation of triglycerides is strong, as a main pathway conserved across taxa.

<table cellspacing="0" class="Table" style="border-collapse:collapse"> <tbody> <tr> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:96px"> Species </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:69px"> Duration </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:109px"> Dose </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:88px"> Increased FA synthesis? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px"> Increased triglyceride? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:108px"> Summary </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px"> Citation </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> Human (Homo sapiens), lab mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> Up to 7 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> 1 μM, 5 μM, and 10 uM T0901317, T0314407 (LXR agonists) for HEK293 cells, 5, 50 mg/kg bdwt T0901317 for mice </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:88px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px"> Increased CYP7A1, SCD-1, and SREBP-1 gene expression vs control in HEK293 cells and C57BL/6 mice, genes linked with fatty acid synthesis, with correlated increases in triglycerides, phospholipids, and HDL cholesterol in a dose-dependent manner. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> Schultz et al. (2000) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> Lab mice (Mus mucsculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 4 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> 10 mg/kg/day T0901317 (LXR agonist) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:88px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px"> Lab mice exposed to 10 mg/kg/day T0901317 had increased gene expression of SRBEP, ACC, FAS, genes linked with fatty acid synthesis, and correlated increased triglycerides, cholesterol, fatty acid. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> Grefhorst et al. (2002) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> Human (Homo sapiens), lab rat (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 96 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> 0.3, 3, 30 nm Insulin plus 2 uM GW3965 (LXR agonist) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:88px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px"> Increased SREBP-1c, FASN, SCD1 gene expression vs control in human and rat cells, with correlated increases in fatty acid synthesis, pointing to increased de novo lipogenesis, in a dose-dependent manner. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> Kotokorpi et al. (2007) </td> </tr> </tbody> </table> In empirical studies, the link between increased fatty acid synthesis and accumulation of triglycerides is generally inferred.   Increased expression of genes and/or signaling molecules known to facilitate fatty acid synthesis, and corresponding increases in triglyceride content in cells, are correlated to show evidence that increases are due to increased synthesis rather than alternative pathways.  Angrish et al. (2016) review genes, signaling molecules, and chemical stressors linked to increased fatty acid synthesis, as well as other pathways leading to accumulation of triglycerides in cells.

Moderate Unspecific

High

Adult

Moderate

Juvenile

Moderate Moderate

Life Stage: 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: Applies to both males and females. Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

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. Grefhorst, A., Elzinga, B.M., Voshol, P.J., Plösch, T., Kok, T., Bloks, V.W., van der Sluijs, F.H., Havekes, L.M., Romijn, J.A., Verkade, H.J., and Kuipers, F.  2002.  Stimulation of Lipogenesis by Pharmacological Activation of the Liver X Receptor Leads to Production of Large, Triglyceride-rich Very Low Density Lipoprotein Particles.  The Journal of Biological Chemistry 277(37): 34182–34190. Kotokorpi, P., Ellis, E., Parini, P., Nilsson, L.-M., Strom, S., Steffensen, K.R., Gustafsson, J.-A., and Mode, A.  2007.  Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965).  Molecular Pharmacology 72(4): 947-955. Landesmann, B., Goumenou, M., Munn, S., and Whelan, M.  2012.  Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity.  European Commission Report EUR 25631, 49 pages.  https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en 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. Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Medina, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B.  2000.  Role of LXRs in control of lipogenesis.  Genes and Development 14:2831–2838.   AOPWiki 2016-11-29T18:41:33

2024-03-29T12:11:15

<upstream-id>4d6be341-6962-45a3-b40e-ef76752deace</upstream-id> <downstream-id>7841b130-5332-458d-a955-36a3916798ca</downstream-id> Increased fatty acid influx is a main pathway broadly accepted as a mechanism for accumulation of triglycerides in cells.  Chemical stressors or alteration of gene expression levels can trigger increased fatty acid influx, as well as changes to membrane permeability and membrane proteins that facilitate fatty acid transport.

The biological plausibility linking increased fatty acid influx to accumulation of triglycerides is strong, as a main pathway conserved across taxa.  Stressors can disrupt normal rates of fatty acid influx, increasing the accumulation of trigylcerides.

In empirical studies, the link between increased fatty acid influx and accumulation of triglycerides is generally inferred.  Zhou et al. (2006) link accumulation of triglycerides to increased fatty acid influx in the livers of transgenic mice with increased Pregnane X Receptor expression compared to wild-type mice.  Increased expression of genes and/or signaling molecules known to facilitate fatty acid influx, and corresponding increases in triglyceride content in cells, are correlated to show evidence that increases are due to increased influx rather than alternative pathways.  Angrish et al. (2016) review genes, signaling molecules, and chemical stressors linked to increased fatty acid influx, as well as other pathways leading to accumulation of triglycerides in cells.  For a review of membrane proteins facilitating fatty acid influx, see Glatz et al. (2010). <table cellspacing="0" class="Table" style="border-collapse:collapse"> <tbody> <tr> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:97px"> Species </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:69px"> Duration </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:110px"> Dose </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:89px"> Increased FA influx? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:71px"> Increased triglyceride? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:109px"> Summary </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:77px"> Citation </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> Lab mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 16 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:110px"> Wild-type versus transgenic-cd36 mice. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:89px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:71px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> Heptatocytes from transgenic-CD36 mice showed increased fatty acid influx than null mice and  measured by the fluorescent fatty acid analog BODIPY and correlated increased triglycerides. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:77px"> Koonen et al. (2007) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> Lab mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:69px"> 1 week, 24 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:110px"> 100 mg/kg/day oral or in vitro 20 um efavirenz </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:89px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:71px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:109px"> Hepatocytes exposed to 20 um efavirenz for 24 hours had increased fatty acid influx as measured by palmitic acid uptake and correlated increased triglycerides and cholesterol to mice exposed to 100 mg/kg/day efavirenz for 1 week. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:77px"> Gwag et al. (2009) </td> </tr> </tbody> </table>

Moderate Unspecific

High

Adult

Moderate

Juvenile

Moderate Moderate

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. Glatz, J.F.C., Joost, J.F., Luiken, P., and Bonen, A.  2010.  Membrane fatty acid transporters as regulators of lipid metabolism: Implications for metabolic disease.  Physiological Reviews 90: 367–417. Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C.  2019.  Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis Journal of Hepatology 70: 930–940. Koonen, D.P.Y., Jacobs, R.L., Febbraio, M. Young, M.E., Soltys, C.-L.M., Ong, H., Vance, D.E., and Dyck, J.R.B.  2007.  Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity.  Diabetes 56: 2863-2871. Landesmann, B., Goumenou, M., Munn, S., and Whelan, M.  2012.  Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity.  European Commission Report EUR 25631, 49 pages.  https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en 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.  The Journal of biological chemistry 281(21): 15013–15020. AOPWiki 2016-11-29T18:41:33

2024-03-29T12:08:16

<upstream-id>7841b130-5332-458d-a955-36a3916798ca</upstream-id> <downstream-id>8a2bca48-23c9-48b7-9af1-4989035b2950</downstream-id>

AOPWiki 2024-03-13T17:57:37

2024-03-13T17:57:37

<upstream-id>536999e0-ec58-4567-aa09-6ec56396a272</upstream-id> <downstream-id>552697e1-19cf-4b16-9139-6e0c62c70a1a</downstream-id> After ligand binding, hepatic PPARγ heterodimerizes with retinoid X receptor and activates target genes involved in lipid storage and metabolism, such as CD36. Subsequently, the CD36 is up-regulated , next the CD36 translocates to the plasma membrane where it can markedly increase the hepatic uptake of fatty acids (FAs) from the circulation.

PPARγ is expressed in the liver (Braissant, Foufelle, Scotto, Dauça, & Wahli, 1996) and regulates CD36 gene transcriptional activation through binding to the peroxisome-proliferator-responsive elements (PPREs) in the promoter region (Teboul et al., 2001). The CD36 is also regulated by several other ligand-sensing and lipogenic transcriptional factors, such as pregnane X receptor, liver X receptor (Zhou et al., 2008) and the aryl hydrocarbon receptor (He, Lee, Febbraio, & Xie, 2011).

Include consideration of temporal concordance here Mice • on high fat diet the levels of PPARγ and CD 36 were increased, additionally inhibition of PPARγ resulted in reduction of CD36 whereas overexpression of receptor lead to overexpression of CD36 protein (Yamazaki, Shiraishi, Kishimoto, Miura, & Ezaki, 2011). • overexpression of PPARγ2 resulted in a marked induction of s PPARγ target gene CD36 in vivo and in vitro in primary hepatocytes (Lee et al., 2012). <table class="prettytable"> <tr> <td> <center>Compound/diet</center> </td> <td> <center>PPARγ activation</center> </td> <td> <center>up-regulation of CD36</center> </td> <td> <center>species</center> </td> <td> <center>Study type</center> </td> <td> <center>Reference</center> </td></tr> <tr> <td> diet rich in saturated fat (fed butter or safflower oil as a high-fat (HF) </td> <td> increase of PPAR γ mRNA (at 4 and 10 weeks) and PPAR γ protein at 4 weeks) </td> <td> mRNA CD36 (at 4 and 10 weeks) </td> <td> C57BL/6J mice </td> <td> In vivo </td> <td> (Yamazaki et al., 2011) </td></tr> <tr> <td> none </td> <td> Overexpression of PPARγ </td> <td> mRNA CD36 </td> <td> C57BL/6 mice/ C3H </td> <td> In vivo </td> <td> (Lee et al., 2012) </td></tr> <tr> <td> none </td> <td> Overexpression of PPARγ </td> <td> mRNA CD36 </td> <td> C57BL/6 mice/ C3H </td> <td> In vitro </td> <td> (Lee et al., 2012) </td></tr> <tr> <td> troglitazone </td> <td> increase of PPAR γ mRNA </td> <td> mRNA CD36 </td> <td> C57BL/6J </td> <td> In vivo </td> <td> (Memon et al., 2000) </td></tr></table> Table 1 Summary of the empirical support for the KER.

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? Contradictory studies have been published investigating the role of PPARγ in the activation of CD36 gene. In contrast to previously reported direct involvement of PPAR in regulation of CD36: Sato et al. suggested an indirect mechanism (Sato, Kuriki, Fukui, & Motojima, 2002).

Braissant, O., Foufelle, F., Scotto, C., Dauça, M., & Wahli, W. (1996). Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology, 137(1), 354–66. He, J., Lee, J. H., Febbraio, M., & Xie, W. (2011). The emerging roles of fatty acid translocase/CD36 and the aryl hydrocarbon receptor in fatty liver disease. Experimental Biology and Medicine (Maywood, N.J.), 236(10), 1116–21. doi:10.1258/ebm.2011.011128 Lee, Y. J., Ko, E. H., Kim, J. E., Kim, E., Lee, H., Choi, H., … Kim, J. (2012). Nuclear receptor PPARγ-regulated monoacylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipid accumulation in diet-induced hepatic steatosis. Proceedings of the National Academy of Sciences of the United States of America, 109(34), 13656–61. doi:10.1073/pnas.1203218109 Memon, R. A., Tecott, L. H., Nonogaki, K., Beigneux, A., Moser, A. H., Grunfeld, C., & Feingold, K. R. (2000). Up-regulation of peroxisome proliferator-activated receptors (PPAR-alpha) and PPAR-gamma messenger ribonucleic acid expression in the liver in murine obesity: troglitazone induces expression of PPAR-gamma-responsive adipose tissue-specific genes in the li. Endocrinology, 141(11), 4021–31. doi:10.1210/endo.141.11.7771 Sato, O., Kuriki, C., Fukui, Y., & Motojima, K. (2002). Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor alpha and gamma ligands. The Journal of Biological Chemistry, 277(18), 15703–11. doi:10.1074/jbc.M110158200 Teboul, L., Febbraio, M., Gaillard, D., Amri, E. Z., Silverstein, R., & Grimaldi, P. A. (2001). Structural and functional characterization of the mouse fatty acid translocase promoter: activation during adipose differentiation. The Biochemical Journal, 360(Pt 2), 305–12. Yamazaki, T., Shiraishi, S., Kishimoto, K., Miura, S., & Ezaki, O. (2011). An increase in liver PPARγ2 is an initial event to induce fatty liver in response to a diet high in butter: PPARγ2 knockdown improves fatty liver induced by high-saturated fat. The Journal of Nutritional Biochemistry, 22(6), 543–53. doi:10.1016/j.jnutbio.2010.04.009 Zhou, J., Febbraio, M., Wada, T., Zhai, Y., Kuruba, R., He, J., … Xie, W. (2008). Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARgamma in promoting steatosis. Gastroenterology, 134(2), 556–67. doi:10.1053/j.gastro.2007.11.037 AOPWiki 2016-11-29T18:41:34

2016-11-29T20:10:52

LXR activation leading to hepatic steatosis

LXR Activation to Liver Steatosis

Marina Goumenou

BY-SA

1.0

Liver steatosis (fatty liver) is considered as one of the first manifestations of possible hepatotoxicity, however it is not regarded as an adverse effect per se and usually can be reversible. The importance of steatosis is highlighted from the fact that it is a prerequisite for the development of non-alcoholic fatty liver disease (NAFLD). NAFLD is a clinicopathological condition that comprises a wide spectrum of liver damage, ranging from steatosis alone to steatohepatitis, advanced fibrosis and cirrhosis. Non-alcoholic steatohepatitis (NASH) represents only a stage in the spectrum of NAFLD and is defined pathologically by the presence of steatosis together with necro-inflammatory activity. The clinical implications of NAFLD are derived mostly from its potential to progress to end-stage liver disease, whereas simple uncomplicated steatosis follows a relatively benign course in most patients. Steatosis is the output of the disturbance on the homeostasis of hepatic lipids which depends on the dynamic balance of several pathways including fatty acid (FA) uptake, de novo FA synthesis, β-oxidation, and very low-density lipoprotein (VLDL) secretion. It is characterized by the accumulation of lipid droplets (mainly triglycerides) in the hepatocytes. This AOP describes the linkage between hepatic steatosis triggered by nuclear receptors activation (PPAR gamma and LXR) through modulation of genes responsible for lipid homeostasis [the carbohydrate response element binding protein (ChREBP), the sterol response element binding protein 1c (SREBP-1c), the free fatty acid uptake transporter FAT/CD36, the fatty acid synthase (FAS), the stearoyl-CoA desaturase 1 (SCD1)] which subsequently leasds to in rease of de novo fatty acids/triglycerides synthesis and fat influx from the peripheral tissues to liver. The accumulation of lipid in the hepatocytes can cause cytoplasm displacement, nucleus distortion, mitochondrial toxicity and eventually necrosis and/or apoptosis. The progression of this condition can lead to tissue inflammation (steatohepatitis) and fibrosis with the involvement of other cells of the hepatic tissue like the Kupffer (inflammation) and the stellate (fibrosis) cells. This purely qualitative AOP description is plausible, the scientific data supporting the AOP are logic, coherent and consistent and there is temporal agreement between the individual KEs. Quantitative data on dose-response-relationships and temporal sequences between key events are still lacking; the provision of quantitative data will strengthen the weight of evidence and make the AOP applicable for chemical risk assessment purposes.

1.1 Binding and activation of receptor PPARγ ligands thiazolidinediones (Rosiglitazone, Pioglitazone, Troglitazone) (Lehmann et al. 1995), (Forman et al. 1995), (Willson et al. 2000). Phthalates MEHP (CAS 4376-20-9) directly binds to PPARγ (Lapinskas et al. 2005), (ToxCastTM Data)in vitro and in silico (Feige et al. 2007), (Rotman et al. 2008), (Kaya et al. 2006) and activates this receptor in transactivation assays (Maloney & Waxman 1999), (Hurst & Waxman 2003b), (Venkata et al. 2006), (ToxCastTM Data). In summary, there is experimental in vitro evidence for binding and transcriptional activation of PPARγ. DEHP (CAS 117-81-7) was not found to bind and activate PPARγ (Lapinskas et al. 2005), (Maloney & Waxman 1999). However recent studies show activation of PPARγ by DEHP(ToxCastTM Data), (Pereira-Fernandes et al. 2013). DEHP was also found to increase the levels of PPARγ in vitro (Lin et al. 2011). Notably, PPARγ is responsive to DEHP in vitro and is translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005). Parabens Butylparaben was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) and mPPARγ in reporter gene assay (Taxvig et al., 2012). Phenols Bisphenol A was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) but not mouse PPARγ (Taxvig et al., 2012) in reporter gene assay. BPA was also reported to increase PPARγ (mRNA) in ovarian granulosa cell line and human luteinized granulosa cells (Kwintkiewicz, Nishi, Yanase, & Giudice, 2010). Organotin Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009). 1.2 Activation of target genes MEHP activation of endogenous PPARγ target genes was evidenced by the stimulation of PPARγ-dependent adipogenesis in the 3T3-L1 cell differentiation model (Hurst & Waxman, 2003).

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Moderate

Consider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP; 6. uncertainties, inconsistencies and data gaps.   <h4>Concordance of dose-response relationships</h4> The existing studies do not provide dose-response curves. However it may be possible in some cases to construct curves from the given numerical data and to relate the dose response for LXR activation with the dose response for TG accumulation in vitro and in vivo in a second more quantitative iteration as the next step of this AOP development.   <h4>Temporal concordance among the key events and adverse outcome</h4> According to the available information the sequence of the events is in strong agreement and consequently the presented MoA could be considered as qualitatively accurate.   <h4>Strength, consistency, and specificity of association of adverse outcome and initiating event</h4> The scientific evidence is presented in Scientific Evidence in support of the MoA.   <h4>Biological plausibility, coherence, and consistency of the experimental evidence</h4> The steatogenic effect of chemicals like LXR ligands is well established in the literature (Peet 1998, Schultz et al. 2000, Horton et al. 2002) and it is well correlated with the expression of the receptor (Moya et al. 2010) the binding to it. In addition it is believed that LXR acts as a cholesterol sensor. Consistent with this role, it has been proposed that LXR induces SREBP-1c in order to generate fatty acids needed for the formation of cholesterol esters, which buffer the free cholesterol concentration (Ferré & Foufelle 2007). Further analysis of the logic, coherence and consistency along with the experimental data has already been presented in Chapter Scientific Evidence in support of the MoA.   <h4>Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP.</h4> As already mentioned abovem there are many other possible MoAs of a chemical in the development of steatosis including MoAs involving the inhibition of β-oxidation, the inhibition of oxidative phosphorylation (leading to a decrease of ATP needed for β-oxidation) and the malfunction of the mechanisms of the excretion of TG from the cell. These pathways are not covered in the presented MoA as they are not directly linked with the activation of LXR. Furthermore, as already explained LXR is not the only receptor which has been identified to be involved in fatty acid metabolism and steatogenesis. Exogenous chemicals acting as ligands for any of the following Nuclear Receptors (AhR, PXR, PPARa, PPARγ and ER) may play a role in the development of steatosis (grey elements on the AOP flow diagram, Fig. 3). There also known interactions or cross-talk between the NRs. Examples of possible interactions are related with fact that LXR is also regulated by the PPARa, the FAT/36 up-regulation from the AhR, PXR and PPARγ, the inhibition of β-oxidation from PPARa and indirectly from the ER. It may be possible from existing literature, or further experimental work to develop MoAs taking binding to each of these receptors as the molecular initiating event and describing the converging pathways leading to steatosis. In fact this work is in progress and indicated as the grey elements on the AOP flow diagram above. The biology of LXR function has been studied using the high affinity synthetic ligands T0901317. According to the study of Mitro (2007), T0901317 binds and activates hPXR and hLXRβ with similar affinity, and can regulate multiple PXR target genes in human cells and mice (like CD36) with similar efficacy to established PXR ligands, but significantly greater potency (Mitro 2007). The author suggested that some of the effects observed with T0901317 such as the more deleterious increase in lipogenesis and hepatic lipid accumulation (in comparison to the LXR-selective GW3965) that have been ascribed to LXR activation maybe the result of simultaneous stimulation of PXR and LXR activity and that the assumption that T0901317 behaves as an LXR-selective agonist may have led to some inaccurate conclusions regarding the effects of LXR activation in vivo. From the data of this study it is evident that SREBP-1c, FAS and SCD-1, which are LXR but not PXR regulated genes, were significantly up-regulated by T0901317. In contrast GW3965 up-regulates less effectively the SREBP-1c, marginally the SCD-1 and not at all the FAS despite the fact that it is considered as a selective LXR agonist (Mitro 2007). The CD36 gene is considered also as a liver specific target of LXR activation (Zhou 2008). However, in the study of Mitro (2007), GW3965 did not up-regulate CD36. These findings could be explained by the lower affinity of this synthetic LXR agonist (EC50 of 0.19 and 0.03 μM for hLXRα and hLXRβ) in relation to the T0901317 (EC50 of 0.02-0.05 μM for both isoforms). Interestingly and despite the low up-regulating activity, GW3965 increases FA and TG accumulation in rat and primary human hepatocytes (Kotokorpi et al. 2007). Based on this information, it could be possible that T0901317 binding on PXR could enhance its steatogenic activity with the proposed MoA still being plausible. This plausibility, however, is clearly related with quantitative aspects. In conclusion, the MoA described can be considered very well supported by the available scientific evidence and it is biologically plausible.   <h4>Uncertainties, inconsistencies and data gaps</h4> The information used for the development of the present pathway is based on in-vitro and in-vivo studies. In the in-vitro studies several cell lines have been used. The expression of the LXR, the SREBP-1c and other elements on these cell lines is a key factor for the plausibility of the pathway in human. According to the study from Moya et al 2010, LXR expression (as measured from mRNA using RT-PCR) in human hepatocytes, HepG2 and HeLa cells was approximately 70%, 70% and 50% in relation to the level of expression in human liver. In addition the expression of SREBP-1c was significantly down-regulated (to less than 25% of normal levels of expression in the liver) in all 3 cell lines. Consequently positive results in relation to fat accumulation after LXR activation from studies using these cell lines may under-estimate the magnitude of effect on human liver while negative results could be interpreted as inconclusive. The assessment of the relative expression of these receptors in other cell lines would be of great importance in order to evaluate the relevance of each in vitro study result. In relation to the in vivo studies which have be made mainly (if not exclusively) in rodents the relevance for humans should be addressed. LXR expression is considered adequately conserved from rodents to humans. In addition it is well known that all the other elements of the pathway are present in human liver. A good example of this is that the well-defined pathogenic role of FAT/CD36 in hepatic steatosis in rodents is also confirmed by the up-regulation in humans of the FAT/CD36 in cases of NASH, NAFLD, insulin resistance, hyperinsulinaemia, HCV and morbidly obese patients (Zhu et al. 2011, Love-Gregory & Abmurad 2011, Miquilena-Colina et al. 2011, Bechmann et al. 2010). However, there is some speculation in relation to the extent that adverse side effects observed in rodents will occur in higher species, including humans. These speculations are raised due to the different behaviour of the LXR agonist GW3965 in in vitro systems which although markedly stimulating lipogenic gene expression in primary human hepatocytes leading to significant TG accumulation at all 3 dose levels after 48 hr, produced only a very modest increase in the triglyceride content in rat cells (Kotokorpi et al. 2007), demonstrating that the use of this rat cell line could underestimate the effect in humans. FA increase was reported in both cell lines. Another interesting finding is that in humans, total CD36 deficiency is relatively common (3–5%) in persons of African and Asian descents (Su & Abmurad 2009). Consequently the presented MoA could be affected mainly quantitatively among humans of different origin. Induction of lipogenic enzymes from the SREBP-1c is evidenced in adult mice but not during the fetal life indicating a different role of the SREBP-1c between these two stages (Liang et al. 2002). This finding gives a strong indication that the presented pathway may be altered in other than adult life stage. Another finding is that of the study of Hu et al. 2005 according to which administration of T0901317 in PPAR-null mice promoted a dose-dependent increase in the rate of peroxisomal β-oxidation in the liver and in relation only to the LXRα. The author suggests that this induction may serve as a counter regulatory mechanism for responding to the hypertriglyceridemia and liver steatosis that is promoted by potent LXR agonists in vivo. T090137 was shown to up-regulate hepatic expression and plasma activity of PLTP in mice in addition to angiopoietin-like protein 3 (Angptl3), playing a critical role in LXR-induced hypertriglyceridemia. However it should be noted that hypertriglyceridemic effect of LXR agonists is usually transient and limited to the first few days of the treatment likely due to enhanced VLDL-triglyceride hydrolysis resulting from increased expression of hepatic LPL (Baranowski 2008). Some studies have demonstrated absence of triglyceride accumulation on SREBP-1c (-/-) mice suggesting that SREBP-1c is a crucial element of the present MoA (Liang et al. 2002, Schultz et al. 2000, Horton et al. 2002, Shimano et al. 1999). In another study in FAT/CD36 knockout mice the effect of LXR agonists on increasing hepatic and circulating levels of triglycerides and free fatty acids (FFAs) was largely abolished suggesting that intact expression and/or activation of FAT/CD36 is required for the steatotic effect of LXR agonists (Febbraio et al. 1999). These two findings together and considering that they are constant and not related with specific experimental conditions could lead one to the hypothesis that both SREBP-1c and CD36 are imperative elements for the cause of steatosis. This hypothesis could be further examined. The present MoA could also be affected by factors related to the formation of steatosis such as trends in adipose tissue (AT) deposition, the total body fat, the visceral AT and the subcutaneous AT which vary among different life stages such as childhood, puberty and adolescence, between sexes and among humans of different origin (Staiano 2012). <h3>Assessment of the quantitative understanding of the AOP</h3> In the present study only qualitative assessment of the proposed MoA was performed. In the studies used there are numerical data mainly to support the expression and up-regulation of the different elements of the pathway. However, further analysis of these numerical data is suggested in following steps. Interestingly, the existence of many network motifs along the pathways was noted during the analysis of the literature, e.g. the positive feed forward LXR up-regulation. This information could be used in the future for the quantitative interpretation of dose response curves and the development of quantitative prediction models of the adverse outcome following the activation of the LXR.

<h2>Confidence in the AOP</h2> Information from this section should be moved to the Key Event Relationship pages! Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains. <h3>How well characterised is the AOP?</h3> Liver steatosis is a well understood adverse outcome. A great number of publications from in vitro, in vivo, mechanistic, clinical and epidemiological studies exist for the qualitative assessment of steatosis. However, the quantitative analysis of the role of a specific exogenous chemical in an adverse outcome in human is a very challenging task due to the involvement of a large number of inter-related factors following the MIE. In fact one chemical may bind to more than one receptor and consequently have different impacts either quantitatively or qualitatively on the downstream events.   <h3>How well are the initiating and other key events causally linked to the outcome?</h3> LXR agonists such T0901317 have been shown to produce LXR activation, as well as triglyceride accumulation, which has been demonstrated in rodent (mouse and rat) and human liver cell lines in vitro. The same chemicals shown to be LXR agonists in the in vitro assays have shown triglyceride accumulation in the liver leading to steatosis in animals and humans through steps of the reported MoA.   <h3>What are the limitations in the evidence in support of the AOP?</h3> Disagreement in the scientific evidence supporting the presented AOP was not found. In relation to data gaps in addition to lack of quantitative information as discussed above there is also lack of specific information in relation to the role of other target genes expressed after the LXR activation.   <h3>Is the AOP specific to certain tissues, life stages / age classes?</h3> There is evidence of different levels of expression of CD36 in different ethnic groups which may be expected to alter the sensitivity to development of steatosis. There may also be differences in expression and role of the same proteins/enzymes in foetal life but this has not been fully elucidated. Further information can be found in the Chapter Uncertainties, inconsistencies and data gaps.   <h3>Are the initiating and key events expected to be conserved across taxa?</h3> From the analysis of the available information from experimental studies using rodents the elements of the MoA appeared to be well conserved between mice and rats. Some concerns in relation to the relevance of the in vivo studies to human are raised mainly due to the different behaviour of the LXR agonist GW3965 which while stimulating lipogenic gene expression in human hepatocytes, causes only a slight increase in TGs in rats (Kotokorpi et al. 2007). Some more differences were also reported between hamsters and monkeys in relation to hypertriglyceridemia (Groot et al. 2005). <h2>Scientific Evidence to Support AOP</h2> Information from this section should be moved to the Key Event Relationship pages! Detailed Description Old format, potemtially to be migrated to table above and underlying articles <table border="1"> <tbody> <tr> <th>Events</th> <th>Scientific Support</th> <th>Strength of Evidence</th> </tr> <tr> <td>LXR binding and activation (Molecular initiating event) receptor </td> <td> <a href="#cite_note-1">[1]</a> <a href="#cite_note-2">[2]</a> <a href="#cite_note-SchultzEtAl2000-3">[3]</a> <a href="#cite_note-4">[4]</a> <a href="#cite_note-5">[5]</a> <a href="#cite_note-6">[6]</a> <a href="#cite_note-7">[7]</a> <a href="#cite_note-8">[8]</a> <a href="#cite_note-9">[9]</a> <a href="#cite_note-10">[10]</a> <a href="#cite_note-Baranowski2008-11">[11]</a> </td> <td>Very Strong</td> </tr> <tr> <td>Binding in the LXREs</td> <td><a href="#cite_note-Baranowski2008-11">[11]</a></td> <td>Very Strong</td> </tr> <tr> <td>Target genes transcription</td> <td><a href="#cite_note-12">[12]</a></td> <td>Very Strong</td> </tr> <tr> <td>Auto-regulation of the LXRa</td> <td> <a href="#cite_note-13">[13]</a> <a href="#cite_note-14">[14]</a> <a href="#cite_note-15">[15]</a> <a href="#cite_note-16">[16]</a> </td> <td>Very Strong</td> </tr> <tr> <td>Increase in expression and activity of the carbohydrate response element-binding protein (ChREBP)</td> <td> <a href="#cite_note-17">[17]</a> <a href="#cite_note-18">[18]</a> </td> <td>Strong According to the study of Denechaud increase in the glucose flux in the cell is a prerequisite for ChREBP activation from T0901317 in mice </td> </tr> <tr> <td>Increase in expression of the SREBP-1c from LXR</td> <td> <a href="#cite_note-19">[19]</a> <a href="#cite_note-SchultzEtAl2000-3">[3]</a> <a href="#cite_note-20">[20]</a> <a href="#cite_note-21">[21]</a> <a href="#cite_note-22">[22]</a> </td> <td>Very Strong However, there are many studies supporting a different behaviour between LXRα and LXRβ, suggesting that SREBP-1c up-regulation is only due to LXRα </td> </tr> <tr> <td>Increase in expression of the SREBP-1c from the ChREBP</td> <td><a href="#cite_note-23">[23]</a></td> <td>Well established</td> </tr> <tr> <td>Induction of lipogenic enzymes from the SREBP-1c (FAS, ACC, GK)</td> <td> <a href="#cite_note-24">[24]</a> <a href="#cite_note-25">[25]</a> <a href="#cite_note-26">[26]</a> <a href="#cite_note-27">[27]</a> <a href="#cite_note-SchultzEtAl2000-3">[3]</a> <a href="#cite_note-28">[28]</a> <a href="#cite_note-29">[29]</a> <a href="#cite_note-30">[30]</a> <a href="#cite_note-31">[31]</a> </td> <td>Very Strong However there is evidence that this effect is not induced in the embryonic state indicating a different role of the SREBP-1c between embryonic and adult life (Liang et al. 2002). It is also suggested that for lipogenic genes, SREBP-1c acts together with ChREBP (Ishii et al. 2004). </td> </tr> <tr> <td>Direct induction of the fatty acid synthase (FAS)</td> <td> <a href="#cite_note-SchultzEtAl2000-3">[3]</a> <a href="#cite_note-32">[32]</a> <a href="#cite_note-33">[33]</a> </td> <td>Very Strong</td> </tr> <tr> <td>Direct induction of the stearoyl-CoA desaturase 1 (SCD1)</td> <td> <a href="#cite_note-34">[34]</a> <a href="#cite_note-35">[35]</a> <a href="#cite_note-SchultzEtAl2000-3">[3]</a> <a href="#cite_note-36">[36]</a> </td> <td>Very Strong</td> </tr> <tr> <td>Up-regulation of the free fatty acid uptake transporter FAT/CD36</td> <td> <a href="#cite_note-37">[37]</a> <a href="#cite_note-38">[38]</a> <a href="#cite_note-39">[39]</a> <a href="#cite_note-40">[40]</a> <a href="#cite_note-41">[41]</a> <a href="#cite_note-42">[42]</a> </td> <td>Very Strong</td> </tr> <tr> <td>De novo fatty acids and triglyceride synthesis</td> <td><a href="#cite_note-43">[43]</a></td> <td>Very Strong</td> </tr> <tr> <td>Fat influx from the peripheral tissues</td> <td> <a href="#cite_note-Baranowski2008-11">[11]</a> <a href="#cite_note-44">[44]</a> <a href="#cite_note-Amacher2011-45">[45]</a> </td> <td>Very Strong</td> </tr> <tr> <td>Steatosis</td> <td> <a href="#cite_note-Amacher2011-45">[45]</a> <a href="#cite_note-46">[46]</a> </td> <td>Very Strong</td> </tr> </tbody> </table> Cite error: <code><ref></code> tags exist, but no <code><references/></code> tag was found AOPWiki 2016-11-29T18:41:16

2024-03-13T17:59:11