Increase, 11β-Hydroxysteroid dehydrogenase type 1 activity

60-35-5

DLFVBJFMPXGRIB-UHFFFAOYSA-N

Acetamide

Acetamid acetamida Acetic acid amide Acetimidic acid Ethanamide Ethanimidic acid Methanecarboxamide NSC 25945

DTXSID7020005

103-90-2

RZVAJINKPMORJF-UHFFFAOYSA-N

Acetaminophen

4-Acetamidophenol APAP Paracetamol 4-hydroxyacetanilide Acetamide, N-(4-hydroxyphenyl)- 4-(Acetylamino)phenol 4-(N-Acetylamino)phenol 4-Acetaminophenol 4'-Hydroxyacetanilide Abensanil Acetagesic Acetalgin ACETAMIDE, N-(4-HYDROXYPHENYL) Acetaminofen Acetanilide, 4'-hydroxy- ACETANILIDE, 4-HYDROXY- Algotropyl Alvedon Anaflon Apamide Banesin Ben-u-ron Bickie-mol Biocetamol Cetadol Citramon P Claratal Clixodyne Dafalgan Daphalgan Dial-a-gesic Disprol Doliprane Dolprone Dymadon Efferalgan Endophy Febrilex Febrilix Febro-Gesic Febrolin Fepanil Finimal Gattaphen T Gelocatil Gutte Enteric Homoolan Jin Gang Lestemp Liquagesic Lonarid Lyteca Syrup Minoset Momentum N-(4-Hydroxyphenyl)acetamide N-Acetyl-4-aminophenol N-Acetyl-4-hydroxyaniline N-Acetyl-p-aminophenol Napafen Naprinol Nobedon NSC 109028 NSC 3991 Ortensan p-(Acetylamino)phenol p-Aceaminophenol Pacemol p-Acetamidophenol p-Acetoaminophen P-ACETYLAMINOPHENOL Paldesic panadeine Panadol Panadol Actifast Panadol Extend Panaleve Panasorb Panodil Paracetamol DC Paracetamole Parageniol Paramol Paraspen Parelan Pasolind N Perfalgan Phenaphen Phendon p-Hydroxyacetanilide Prodafalgan Puerxitong Pyrinazine Resfenol Resprin Rhodapop NCR Salzone Tabalgin Tachipirina Tempanal Tralgon Tylenol TylolHot Valadol Valgesic Vermidon Vick Pyrena

DTXSID2020006

968-81-0

VGZSUPCWNCWDAN-UHFFFAOYSA-N

Acetohexamide

Benzenesulfonamide, 4-acetyl-N-[(cyclohexylamino)carbonyl]- 1-(p-Acetylbenzenesulfonyl)-3-cyclohexylurea 1-[(p-Acetylphenyl)sulfonyl]-3-cyclohexylurea Acetohexamid acetohexamida Dimelin Dimelor Dymelor Gamadiabet Hypoglicil Metaglucina Minoral N-(p-Acetylphenylsulfonyl)-N'-cyclohexylurea Ordimel Tsiklamid Urea, 1-[(p-acetylphenyl)sulfonyl]-3-cyclohexyl-

DTXSID7020007

67-66-3

HEDRZPFGACZZDS-UHFFFAOYSA-N

Chloroform

Trichloromethane Methane, trichloro- CARBON TRICHLORIDE Chloroforme cloroformo Formyl trichloride Methane trichloride Methane,trichloro- NSC 77361 Trichloroform UN 1888

DTXSID1020306

110-00-9

YLQBMQCUIZJEEH-UHFFFAOYSA-N

Furan

Divinylene oxide furanne Furfuran Oxacyclopentadiene Tetrole UN 2389

DTXSID6020646

7429-90-5

XAGFODPZIPBFFR-UHFFFAOYSA-N

AZDRQVAHHNSJOQ-UHFFFAOYSA-N

Aluminum

Aisin Metal Fiber Al 050P-H24 ALC Fine Alcan XI 1391 Almi-Paste SSP 303AR Aloxal 3010 Alpaste 00-0506 Alpaste 0100M Alpaste 0100MA Alpaste 0100M-C Alpaste 0200M Alpaste 0200T Alpaste 0230M Alpaste 0230T Alpaste 0241M Alpaste 0300M Alpaste 0500M Alpaste 0539X Alpaste 0620MS Alpaste 0625TS Alpaste 0638-70C Alpaste 0700M Alpaste 0780M Alpaste 0900M Alpaste 100M Alpaste 100MS Alpaste 100MSR Alpaste 1100M Alpaste 1100MA Alpaste 1100N Alpaste 1100NA Alpaste 1109MA Alpaste 1109MC Alpaste 1200M Alpaste 1200T Alpaste 1260MS Alpaste 1500MA Alpaste 1700NL Alpaste 1810YL Alpaste 1830YL Alpaste 1900M Alpaste 1900XS Alpaste 1950M Alpaste 1950N Alpaste 210N Alpaste 2172EA Alpaste 2173 Alpaste 240T Alpaste 241M Alpaste 417 Alpaste 46-046 Alpaste 4-621 Alpaste 4919 Alpaste 50-63 Alpaste 50-635 Alpaste 51-148B Alpaste 51-231 Alpaste 5205N Alpaste 5207N Alpaste 52-509 Alpaste 52-568 Alpaste 5301N Alpaste 5302N Alpaste 53-119 Alpaste 5422NS Alpaste 54-452 Alpaste 54-497 Alpaste 54-542 Alpaste 55-516 Alpaste 55-519 Alpaste 55-574 Alpaste 5620NS Alpaste 5630NS Alpaste 5640NS Alpaste 56-501 Alpaste 5650NS Alpaste 5653NS Alpaste 5654NS Alpaste 5680N Alpaste 5680NS Alpaste 60-600 Alpaste 60-760 Alpaste 60-768 Alpaste 62-356 Alpaste 6340NS Alpaste 6370NS Alpaste 6390NS Alpaste 640NS Alpaste 65-388 Alpaste 66NLB Alpaste 710N Alpaste 7130N Alpaste 7160N Alpaste 7160NS Alpaste 725N Alpaste 740NS Alpaste 7430NS Alpaste 7580NS Alpaste 7620NS Alpaste 7640NS Alpaste 7670M Alpaste 7670NS Alpaste 7675NS Alpaste 7679NS Alpaste 7680N Alpaste 7680NS Alpaste 76840NS Alpaste 7730N Alpaste 7770N Alpaste 7830N Alpaste 8004 Alpaste 8080N Alpaste 8260NAR Alpaste 891K Alpaste 91-0562 Alpaste 92-0592 Alpaste 93-0595 Alpaste 93-0647 Alpaste 94-2315 Alpaste 95-0570 Alpaste 96-0635 Alpaste 96-2104 Alpaste 97-0510 Alpaste 97-0534 Alpaste AW 520B Alpaste AW 612 Alpaste AW 9800 Alpaste F 795 Alpaste FM 7680K Alpaste FX 440 Alpaste FX 910 Alpaste FZ 0534 Alpaste FZU 40C Alpaste G Alpaste HR 8801 Alpaste HS 2 Alpaste J Alpaste K 9800 Alpaste MC 666 Alpaste MC 707 Alpaste MF 20 Alpaste MG 01 Alpaste MG 1000 Alpaste MG 1300 Alpaste MG 500 Alpaste MG 600 Alpaste MH 6601 Alpaste MH 8801 Alpaste MH 9901 Alpaste MR 7000 Alpaste MR 9000 Alpaste MS 630 Alpaste N 1700NL Alpaste NS 7670 Alpaste O 100N Alpaste O 2130 Alpaste O 300M Alpaste P 0100 Alpaste P 1950 Alpaste S Alpaste SAP 110 Alpaste SAP 414P Alpaste SAP 550N Alpaste SCR 5070 Alpaste TCR 2020 Alpaste TCR 2060 Alpaste TCR 2070 Alpaste TCR 3010 Alpaste TCR 3030 Alpaste TCR 3040 Alpaste TCR 3130 Alpaste TD 200T Alpaste UF 500 Alpaste WB 0230 Alpaste WD 500 Alpaste WJP-U 75C Alpaste WX 0630 Alpaste WX 7830 Alpaste WXA 7640 Alpaste WXM 0630 Alpaste WXM 0650 Alpaste WXM 0660 Alpaste WXM 1415 Alpaste WXM 1440 Alpaste WXM 5422 Alpaste WXM 760b Alpaste WXM 7640 Alpaste WXM 7675 Alpaste WXM-T 60B Alpaste WXM-U 75 Alpaste WXM-U 75C Altop X Aluchrome Ultrafin Super Alumat 1600 Alumet H 30 aluminio Aluminium Aluminium Flake Aluminum 27 Aluminum atom Aluminum element Aluminum Flake PCF 7620 Aluminum granules ALUMINUM METAL/GRANULE ALUMINUM PASTE ALUMINUM PIGMENT ALUMINUM TURNINGS Alumi-paste 640NS Alumipaste 91-0562 Alumipaste 98-1822T Alumipaste AW 620 Alumipaste CR 300 Alumipaste GX 180A Alumipaste GX 201A Alumipaste HR 7000 Alumipaste HR 850 Alumipaste MG 11 Alumipaste MH 8801 Aquamet NPW 2900 Aquapaste 205-5 Aquasilver LPW Astroflake 40 Astroflake Black N 020 Astroflake Black N 070 Astroflake LG 40 Astroflake LG 70 Astroflake Silver N 040 Astroshine NJ 1600 Astroshine T 8990 Atomizalumi VA 200 C.I. PIGMENT METAL 1 Chromal IV Chromal X Decomet 1001/10 Decomet 2018/10 Decomet High Gloss Al 1002/10 Ecka AS 081 Eckart 9155 Eterna Brite 301-1 Eterna Brite 601-1 Eterna Brite 651-1 Eterna Brite EBP 251PA Eterna Brite Primier 251PA Ferro FX 53-038 Friend Color F 500GR-W Friend Color F 500WT Friend Color F 700RE-W Friend Color F 701RE-W Hi Print 60T High Print 60T Hisparkle HS 2 Hydro Paste 8726 Hydrolac WHH 2153 Hydrolan 3560 Hydrolux Reflexal 100 Hydroshine WS 1001 JISA 51010P Kryal Z Lansford 243 LE Sheet 800 Leafing Alpaste LG-H Silver 25 Lunar Al-V 95 Metallux 161 Metallux 2154 Metallux 2192 Metalure Metalure 55350 Metalure L 55350 Metalure L 59510 Metalure W 2001 Metapor Metasheen 1800 Metasheen HR 0800 Metasheen KM 100 Metasheen KM 1000 Metasheen Slurry 1807 Metasheen Slurry 1811 Metasheen Slurry KM 100 Metax G Metax S Mirror Glow 1000 Mirror Glow 600 Mirrorsheen Noral Aluminium Noral Ink Grade Aluminium Obron 10890 Offset FM 4500 Puratronic Reflexal 145 Reynolds 400 Reynolds 4-301 Reynolds 4-591 Reynolds 667 SAP 260PW-HS SAP-FM 4010 SBC 516-20Z Scotchcal 7755SE Serumekku Setanium 50MIS-H8 Siberline ET 2025 Siberline ST 21030E1 Silvar A Silver VT 522 Silverline SSP 353 Silvex 793-20C Sparkle Silver 3141ST Sparkle Silver 3500 Sparkle Silver 3641 Sparkle Silver 5000AR Sparkle Silver 516AR Sparkle Silver 5242AR Sparkle Silver 5245AR Sparkle Silver 5271AR Sparkle Silver 5500 Sparkle Silver 5745 Sparkle Silver 7000AR Sparkle Silver 7005AR Sparkle Silver 7500 Sparkle Silver 960-25E1 Sparkle Silver E 1745AR Sparkle Silver L 1526AR Sparkle Silver Premier 751 Sparkle Silver SS 3130 Sparkle Silver SS 5242AR Sparkle Silver SS 5588 Sparkle Silver SSP 132AR Special PCR 507 Splendal 6001BG Spota Mobil 801 SSP 760-20C Stapa Aloxal PM 2010 Stapa Aloxal PM 3010 Stapa Aloxal PM 4010 Stapa Hydrolac BG 8n.1 Stapa Hydrolac BGH Chromal X Stapa Hydrolac PM Chromal VIII Stapa Hydrolac W 60NL Stapa Hydrolac WH 16 Stapa Hydrolac WH 66NL Stapa Hydrolux 2192 Stapa Hydrolux 8154 Stapa IL Hydrolan 2192-55900G Stapa Metallic R 607 Stapa Metallux 1050 Stapa Metallux 211 Stapa Metallux 212 Stapa Metallux 2196 Stapa Metallux 274 Stapa Mobilux 181 Stapa Offset 3000 Stapa PV 10 Stapa VP 46432G Starbrite 2100 Super Fine 18000 Super Fine 22000 Supramex 2022 Toyo Aluminum 02-0005 Toyo Aluminum 93-3040 Transmet K 102HE Tufflake 3645 Tufflake 5843 UN 1396 US Aluminum 809 Valimet H 2 Valimet H 3 White Silver 7080N White Silver 7130N

DTXSID3040273

7440-43-9

BDOSMKKIYDKNTQ-UHFFFAOYSA-N

Cadmium

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

DTXSID1023940

7439-97-6

QSHDDOUJBYECFT-UHFFFAOYSA-N

Mercury

Liquid silver Mercure MERCURIC METAL TRIPLE DISTILLED mercurio Mercury element Quecksilber Quicksilver UN 2024 UN 2809

DTXSID1024172

7440-61-1

JFALSRSLKYAFGM-UHFFFAOYSA-N

Uranium

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

DTXSID1042522

7440-38-2

RQNWIZPPADIBDY-UHFFFAOYSA-N

Arsenic

As Arsenic black ARSENIC METAL arsenico Grey arsenic UN 1558

DTXSID4023886

7440-22-4

BQCADISMDOOEFD-UHFFFAOYSA-N

Silver

Ag Nanopaste NPS-J 90 Ag Sphere 2 Ag-C-GS Algaedyn Arctic Silver 3 Argentum Astroflake 5 Carey Lea silver Colloidal silver Dotite XA 208 Du Pont 4943 ECM 100AF4810 Enlight 600 Enlight silver plate 600 Epinall Finesphere SVND 102 Fordel DC FP 5369-502 Jelcon SH 1 Jungindai Takasago 300 KS (metal) LCP 1-19SFS Metz 3000-1 Nanomelt AGC-A Nanomelt Ag-XA 301 Nanomelt Ag-XF 301 Nanomelt Ag-XF 301H Nanopaste NPS-J 90 Perfect Silver Puff Silver X 1200 RT 1710S-C1 SD (metal) Shell Silver Silbest E 20 Silbest F 20 Silbest J 18 Silbest TC 12 Silbest TC 20E Silbest TC 25A Silbest TCG 1 Silbest TCG 7 Silcoat AgC 103 Silcoat AgC 2011 Silcoat AgC 209 Silcoat AgC 2190 Silcoat AgC 222 Silcoat AgC 2411 Silcoat AgC 74T Silcoat AgC-A Silcoat AgC-AO Silcoat AgC-B Silcoat AgC-BO Silcoat AgC-D Silcoat AgC-G Silcoat AgC-GS Silcoat AgC-L Silcoat AgC-O Silcoat GS Silcoat RF 200 Silflake 135 Silsphere 514 Silver atom Silver element Silver Flake 1 Silver Flake 25 Silver Flake 52 Silver Flake 7A SILVER FLAKES Silver metal Silvest TCG 11N Technic 299 Technic 450 Techno Alpha 175

DTXSID4024305

7439-96-5

PWHULOQIROXLJO-UHFFFAOYSA-N

Manganese

Colloidal manganese Cutaval Manganese element Manganese fulleride Manganese metal alloy Manganese-55 manganeso

DTXSID2024169

7440-02-0

PXHVJJICTQNCMI-UHFFFAOYSA-N

Nickel

Carbonyl 255 Carbonyl Ni 123 Carbonyl Ni 283 Carbonyl Nickel 123 Carbonyl Nickel 283 Carbonyl Nickel 287 Cerac N 2003 CNS 10 Micron Exmet 4 Ni X-4/0 Fibrex P Incofoam Nickel element NICKEL ROUND ANODES Nicrobraz LM:BNi 2 Ni-Flake 95 Novamet 123 Novamet 4SP Novamet 4SP10 Novamet 525 Novamet CNS 400 Novamet HCA 1 Novamet NI 255 Raney nickel Raney nickel 2800 UN 1325 UN 2881

DTXSID2020925

7440-66-6

HCHKCACWOHOZIP-UHFFFAOYSA-N

Zinc

Zn Asarco L 15 C.I. Pigment Black 16 Merrillite NC-Zinc Rheinzink Stapa TE Zinc AT UF (metal) UN 1436 Zinc dust Zinc Dust 3 Zinc Dust 500 mesh Zinc Dust LS 2 Zinc Dust MCS Zinc Flakes GTT ZINC METAL ZINC MOSSY ZINC STRIP ZINC, MOSSY Zincsalt GTT

DTXSID7035012

PR:000011406

PR glucocorticoid receptor

CL:0000091

CL Kupffer cell

CL:0000632

CL hepatic stellate cell

CHEBI:3815

CHEBI collagen

UBERON:0002107

UBERON liver

GO:0004883

GO glucocorticoid receptor activity

HP:0001397

HP Hepatic steatosis

MP:0003674

MP oxidative stress

GO:0008219

GO cell death

GO:0042116

GO macrophage activation

GO:0035733

GO hepatic stellate cell activation

GO:0032964

GO collagen biosynthetic process

MP:0003333

MP liver fibrosis

WIKI increased

WIKI occurrence

Acetaminophen

2016-11-29T18:42:26

Chloroform

2016-11-29T18:42:27

furan

2020-05-01T14:35:22

Platinum

2022-02-04T14:36:54

Aluminum

2022-02-04T14:42:11

Cadmium

2017-10-25T08:33:12

Mercury

2016-11-29T18:42:19

Uranium

2021-08-05T14:28:50

Arsenic

2021-04-27T00:15:21

Silver

2022-02-03T11:20:11

Manganese

2022-02-04T14:47:23

Nickel

2022-02-04T14:47:59

Zinc

2022-02-04T15:05:00

nanoparticles

2016-12-21T09:40:06

WikiUser_28

Vertebrates

WikiUser_26

ApacheUser rodents

9606

NCBI Homo sapiens

WCS_9606

common toxicological species human

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

10116

NCBI Rattus norvegicus

10090

NCBI mouse

9823

NCBI pigs

10116

NCBI rat

WikiUser_17

mammals

7955

NCBI zebra fish

36500

NCBI salmonid fish

Increase, 11β-Hydroxysteroid dehydrogenase type 1 activity

Increase, 11β-HSD1 activity

Molecular

AOPWiki 2026-02-18T08:38:26

2026-02-18T08:38:26

Increase, Hepatic intracellular active glucocorticoids

Increase, Hepatic intracellular active GC

Cellular

AOPWiki 2026-02-18T08:43:05

2026-02-26T06:48:35

Increase, Glucocorticoid receptor activation

Increase, GR activation

Molecular

Site of action:  The molecular site of action is the glucocorticoid receptor (GR), nuclear receptor part of a superfamily of highly conserved which bind to steroids, sterols, thyroid hormones, retinoids, and orphan receptors (Weikum et al., 2017). In humans, the formal gene name of this receptor is nuclear receptor subfamily 3, group C, member 1 – NR3C1 (Oakley & Cidlowski, 2013). More specifically, the GR agonism occurs through the interaction of a chemical (endogenous compounds such as cortisol, or an external stressor) with the ligand binding domain. In the absence of a ligand, the GR is transcriptionally inactive in the cytoplasm (Barnes, 1998). Responses at the macromolecular level:  Once bound to a hormonal ligand, the GR is translocated from the cytoplasm to the nucleus where the activated GR interacts with genomic glucocorticoid-response elements (GRE) and regulates transcription of associated genes. Interactions with double stranded DNA and transcription factors can cause both activation and repression of downstream genes via directly binding to a consensus site, binding to other transcription factors to form a heterodimer, or homodimerization prior to DNA binding (Oakley & Cidlowski, 2013).

Glucocorticoid receptor activation can be measured via bioanalytical tools such as in vitro bioassays where results are typically reported in Dexamethasone-equivalents (DEX-EQ) . However it should be noted that these assays have differences in sensitivity (Cole & Brooks, 2023).             <table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <caption>In Vitro Assays Employed in Glucocorticoid Receptor Agonism Detection</caption> <tbody> <tr> <td>Assay</td> <td>Receptor Organism</td> <td>Tissue</td> <td>Citation</td> </tr> <tr> <td> TOX21 GR BLA Agonist Ratio </td> <td>Human</td> <td>Cervix</td> <td>Huang et al., 2011</td> </tr> <tr> <td>GR CALUX</td> <td>Human</td> <td>Osteosarcoma</td> <td>Been et al., 2021; Macikova et al., 2014; Schriks et al., 2010; Suzuki et al., 2015</td> </tr> <tr> <td>Attagene GR TRANS</td> <td>Human</td> <td>Liver</td> <td>Martin et al., 2010; Medvedev et al., 2018; Romanov et al., 2008</td> </tr> <tr> <td>Attagene GRe CIS</td> <td>Human</td> <td>Liver</td> <td>Martin et al., 2010; Medvedev et al., 2018; Romanov et al., 2008</td> </tr> <tr> <td>CV1-hGR</td> <td>Human</td> <td>Kidney</td> <td>Medlock Kakaley et al., 2019</td> </tr> <tr> <td>GR-GeneBlazer</td> <td>Human</td> <td>Kidney</td> <td>Jia et al., 2016</td> </tr> <tr> <td>NovaScreen NR hGR</td> <td>Human</td> <td>N/A</td> <td>Knudsen et al., 2011; Sipes et al., 2013</td> </tr> <tr> <td>Trout GR1</td> <td>Trout</td> <td>Kidney</td> <td>Kugathas & Sumpter, 2011</td> </tr> <tr> <td>Trout GR2</td> <td>Trout</td> <td>Kidney</td> <td>Kugathas & Sumpter, 2011</td> </tr> <tr> <td>Indigo hGR</td> <td>Human</td> <td>N/A</td> <td>Cavaillin et al., 2021; Cole et al., 2025</td> </tr> <tr> <td>Indigo zfGR</td> <td>Zebrafish</td> <td>N/A</td> <td>Cole et al., 2025</td> </tr> </tbody> </table>   In addition to bioanalytical techniques, induction of GR-regulated genes are also indicative of GR agonism in vivo (Cavallin et al., 2021; Cole et al., 2025; Garland et al., 2019).

Taxonomic Applicability: The GR is present in almost every vertebrate cell (Weikum et al., 2017). The evolutionary conservation of GR activation across taxa was examined in silico through the employment of EPA’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) Tool, and 623 orthologs were identified confirming conservation in vertebrate species. Additionally, bioanalytical methods comparing zebrafish (Danio rerio) GR and human GR show conservation of ligand binding and receptor agonism when using dexamethasone and beclomethasone dipropionate. Lastly, the fathead minnow (Pimephales promelas) model has been employed to examine susceptibility to synthetic glucocorticoids in the following in vivo exposure to dexamethasone and beclomethasone dipropionate (Cole et al., 2025). Through the processes of gene duplication and divergence, the GR and mineralocorticoid receptor (MR) evolved from a corticoid receptor in jawless fish. While only possessing one isoform of MR, teleost fish possess two isoforms of the GR and all three have affinity for endogenous cortisol (Baker et al., 2013). Conservation of susceptibility does not infer similarities in sensitivity which varies based on species, receptor isoform, and tissue (Aedo et al., 2023; Baker et al., 2013; Bury & Sturm, 2007; Gilmour, 2005; Jerez-Cepa et al., 2019; Small & Quiniou, 2018; Stolte et al., 2006) <img alt="Results from (A) Level 1 Sequence Alignment to Predict Cross-Species Susceptibility (SeqAPASS) comparing 1,631 protein sequences to zebrafish glucocorticoid receptor (zfGR). Analysis resulted in 782 ortholog candidates at a susceptibility cut-off of 20.55%. (B) Level 2 SeqAPASS analysis examining the ligand binding domain (LDB) of zfGR which resulted in 784 orthologs at a susceptibility cut-off of 34.47%." src="https://aopwiki.org/system/dragonfly/production/2025/04/18/9oyje58ylm_image_5_.png" style="height:1745px; width:1505px" /> Figure: Results from (A) Level 1 Sequence Alignment to Predict Cross-Species Susceptibility (SeqAPASS) comparing 1,631 protein sequences to zebrafish glucocorticoid receptor (zfGR). Analysis resulted in 782 ortholog candidates at a susceptibility cut-off of 20.55%. (B) Level 2 SeqAPASS analysis examining the ligand binding domain (LDB) of zfGR which resulted in 784 orthologs at a susceptibility cut-off of 34.47%.   Life Stage Applicability: This MIE is not life stage specific. However, the downstream transcriptional effects of GR agonism may vary based on life stage. (LaLone et al., 2012; Watanabe et al., 2016).   Sex Applicability: This MIE is not sex specific.

CL:0000255

CL eukaryotic cell

High Unspecific

Moderate

All life stages

High

Aedo, J. E., Zuloaga, R., Aravena-Canales, D., Molina, A., & Valdés, J. A. (2023). Role of glucocorticoid and mineralocorticoid receptors in rainbow trout (Oncorhynchus mykiss) skeletal muscle: A transcriptomic perspective of cortisol action. Frontiers in Physiology, 13, 1048008. https://doi.org/10.3389/fphys.2022.1048008 Baker, M. E., Funder, J. W., & Kattoula, S. R. (2013). Evolution of hormone selectivity in glucocorticoid and mineralocorticoid receptors. The Journal of Steroid Biochemistry and Molecular Biology, 137, 57–70. Barnes, P. J. (1998). Anti-inflammatory actions of glucocorticoids: Molecular mechanisms. Clinical Science (London, England: 1979), 94(6), 557–572. https://doi.org/10.1042/cs0940557 Been, F., Pronk, T., Louisse, J., Houtman, C., van der Velden-Slootweg, T., van der Oost, R., & Dingemans, M. M. L. (2021). Development of a framework to derive effect-based trigger values to interpret CALUX data for drinking water quality. Water Research, 193. https://doi.org/10.1016/j.watres.2021.116859 Bury, N. R., & Sturm, A. (2007). Evolution of the corticosteroid receptor signalling pathway in fish. General and Comparative Endocrinology, 153(1), 47–56. https://doi.org/10.1016/j.ygcen.2007.03.009 Cavallin, J. E., Battaglin, W. A., Beihoffer, J., Blackwell, B. R., Bradley, P. M., Cole, A. R., Ekman, D. R., Hofer, R. N., Kinsey, J., Keteles, K., Weissinger, R., Winkelman, D. L., & Villeneuve, D. L. (2021). Effects-Based Monitoring of Bioactive Chemicals Discharged to the Colorado River before and after a Municipal Wastewater Treatment Plant Replacement. Environmental Science & Technology, 55(2), 974–984. https://doi.org/10.1021/acs.est.0c05269 Cole, A. R., & Brooks, B. W. (2023). Comparative Endpoint Sensitivity of Bioanalytical Tools for Glucocorticoid Receptor Agonism Surveillance in Aquatic Matrices. ACS ES&T Water, 3(9), 3082–3092. <a href="https://doi.org/10.1021/acsestwater.3c00253">https://doi.org/10.1021/acsestwater.3c00253</a> Cole, A. R., Blackwell, B. R., Cavallin, J. E., Collins, J. E., Kittelson, A. R., Shmaitelly, Y. M., Langan, L. M., Villenueve, D. L., & Brooks, B. W. (2025). Comparative Glucocorticoid Receptor Agonism: In Silico, In Vitro, and In Vivo and Identification of Potential Biomarkers for Synthetic Glucocorticoid Exposure. Environmental Toxicology and Chemistry, vgae041. https://doi.org/10.1093/etojnl/vgae041 Garland, M. A., Sengupta, S., Mathew, L. K., Truong, L., de Jong, E., Piersma, A. H., La Du, J., & Tanguay, R. L. (2019). Glucocorticoid receptor-dependent induction of cripto-1 (one-eyed pinhead) inhibits zebrafish caudal fin regeneration. Toxicology Reports, 6, 529–537. https://doi.org/10.1016/j.toxrep.2019.05.013 Gilmour, K. M. (2005). Mineralocorticoid receptors and hormones: Fishing for answers. Endocrinology, 146(1), 44–46. Huang, R., Xia, M., Cho, M.-H., Sakamuru, S., Shinn, P., Houck, K. A., Dix, D. J., Judson, R. S., Witt, K. L., Kavlock, R. J., Tice, R. R., & Austin, C. P. (2011). Chemical Genomics Profiling of Environmental Chemical Modulation of Human Nuclear Receptors. Environmental Health Perspectives, 119(8), 1142–1148. https://doi.org/10.1289/ehp.1002952 Jerez-Cepa, I., Gorissen, M., Mancera, J. M., & Ruiz-Jarabo, I. (2019). What can we learn from glucocorticoid administration in fish? Effects of cortisol and dexamethasone on intermediary metabolism of gilthead seabream (Sparus aurata L.). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 231, 1–10. https://doi.org/10.1016/j.cbpa.2019.01.010 Jia, A., Wu, S., Daniels, K. D., & Snyder, S. A. (2016). Balancing the Budget: Accounting for Glucocorticoid Bioactivity and Fate during Water Treatment. Environmental Science & Technology, 50(6), 2870–2880. https://doi.org/10.1021/acs.est.5b04893 Knudsen, T. B., Houck, K. A., Sipes, N. S., Singh, A. V., Judson, R. S., Martin, M. T., Weissman, A., Kleinstreuer, N. C., Mortensen, H. M., Reif, D. M., Rabinowitz, J. R., Setzer, R. W., Richard, A. M., Dix, D. J., & Kavlock, R. J. (2011). Activity profiles of 309 ToxCastTM chemicals evaluated across 292 biochemical targets. Toxicology, 282(1), 1–15. https://doi.org/10.1016/j.tox.2010.12.010 Kugathas, S., & Sumpter, J. P. (2011). Synthetic Glucocorticoids in the Environment: First Results on Their Potential Impacts on Fish. Environmental Science & Technology, 45(6), 2377–2383. https://doi.org/10.1021/es104105e LaLone, C. A., Villeneuve, D. L., Olmstead, A. W., Medlock, E. K., Kahl, M. D., Jensen, K. M., Durhan, E. J., Makynen, E. A., Blanksma, C. A., Cavallin, J. E., Thomas, L. M., Seidl, S. M., Skolness, S. Y., Wehmas, L. C., Johnson, R. D., & Ankley, G. T. (2012). Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development. Environmental Toxicology and Chemistry, 31(3), 611–622. https://doi.org/10.1002/etc.1729 Macikova, P., Groh, K. J., Ammann, A. A., Schirmer, K., & Suter, M. J.-F. (2014). Endocrine Disrupting Compounds Affecting Corticosteroid Signaling Pathways in Czech and Swiss Waters: Potential Impact on Fish. Environmental Science & Technology, 48(21), 12902–12911. https://doi.org/10.1021/es502711c Martin, M. T., Dix, D. J., Judson, R. S., Kavlock, R. J., Reif, D. M., Richard, A. M., Rotroff, D. M., Romanov, S., Medvedev, A., Poltoratskaya, N., Gambarian, M., Moeser, M., Makarov, S. S., & Houck, K. A. (2010). Impact of Environmental Chemicals on Key Transcription Regulators and Correlation to Toxicity End Points within EPA’s ToxCast Program. Chemical Research in Toxicology, 23(3), 578–590. https://doi.org/10.1021/tx900325g Medlock Kakaley, E., Cardon, M. C., Gray, L. E., Hartig, P. C., & Wilson, V. S. (2019). Generalized Concentration Addition Model Predicts Glucocorticoid Activity Bioassay Responses to Environmentally Detected Receptor-Ligand Mixtures. Toxicological Sciences, 168(1), 252–263. https://doi.org/10.1093/toxsci/kfy290 Medvedev, A., Moeser, M., Medvedeva, L., Martsen, E., Granick, A., Raines, L., Zeng, M., Makarov, S., Houck, K. A., & Makarov, S. S. (2018). Evaluating biological activity of compounds by transcription factor activity profiling. Science Advances, 4(9), eaar4666. https://doi.org/10.1126/sciadv.aar4666 Oakley, R. H., & Cidlowski, J. A. (2013). The Biology of the Glucocorticoid Receptor: New Signaling Mechanisms in Health and Disease. The Journal of Allergy and Clinical Immunology, 132(5), 1033–1044. https://doi.org/10.1016/j.jaci.2013.09.007 Romanov, S., Medvedev, A., Gambarian, M., Poltoratskaya, N., Moeser, M., Medvedeva, L., Gambarian, M., Diatchenko, L., & Makarov, S. (2008). Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nature Methods, 5(3), 253–260. https://doi.org/10.1038/nmeth.1186 Schriks, M., van Leerdam, J. A., van der Linden, S. C., van der Burg, B., van Wezel, A. P., & de Voogt, P. (2010). High-Resolution Mass Spectrometric Identification and Quantification of Glucocorticoid Compounds in Various Wastewaters in The Netherlands. Environmental Science & Technology, 44(12), 4766–4774. https://doi.org/10.1021/es100013x Sipes, N. S., Martin, M. T., Kothiya, P., Reif, D. M., Judson, R. S., Richard, A. M., Houck, K. A., Dix, D. J., Kavlock, R. J., & Knudsen, T. B. (2013). Profiling 976 ToxCast Chemicals across 331 Enzymatic and Receptor Signaling Assays. Chemical Research in Toxicology, 26(6), 878–895. https://doi.org/10.1021/tx400021f Small, B. C., & Quiniou, S. M. A. (2018). Characterization of two channel catfish, Ictalurus punctatus, glucocorticoid receptors and expression following an acute stressor. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 216, 42–51. https://doi.org/10.1016/j.cbpa.2017.11.011 Stolte, E. H., Kemenade, B. M. L. V. van, Savelkoul, H. F. J., & Flik, G. (2006). Evolution of glucocorticoid receptors with different glucocorticoid sensitivity. Journal of Endocrinology, 190(1), 17–28. https://doi.org/10.1677/joe.1.06703 Suzuki, G., Sato, K., Isobe, T., Takigami, H., Brouwer, A., & Nakayama, K. (2015). Detection of glucocorticoid receptor agonists in effluents from sewage treatment plants in Japan. Science of The Total Environment, 527–528, 328–334. https://doi.org/10.1016/j.scitotenv.2015.05.008 Watanabe, Y., Grommen, S. V. H., & De Groef, B. (2016). Corticotropin-releasing hormone: Mediator of vertebrate life stage transitions? General and Comparative Endocrinology, 228, 60–68. https://doi.org/10.1016/j.ygcen.2016.02.012 Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: Precision and plasticity via allostery. Nature Reviews Molecular Cell Biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152   AOPWiki 2016-11-29T18:41:22

2026-02-12T07:24:33

Increase, De novo lipogenesis

Cellular

UBERON:0002107

UBERON liver

CL:0000182

CL hepatocyte

AOPWiki 2017-04-12T14:46:28

2026-02-10T04:39:07

Increase, Liver steatosis

Organ

Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes.   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).  Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes. Role in biology: steatosis is an adverse endpoint.  Consequences: Liver steatosis, or fatty liver, serves as a pivotal factor in the development of liver fibrosis by triggering a cascade of pathological events. According to the two-strikes hypothesis (Day and James, 1998), liver damage progresses in two stages: the first strike involves the accumulation of lipids in hepatocytes, often due to metabolic disturbances such as insulin resistance, excess free fatty acids, or oxidative stress. This stage, though asymptomatic, increases liver vulnerability by inducing mild oxidative stress and inflammation. The second strike introduces additional insults, such as inflammatory mediators or cellular damage, exacerbating liver injury and promoting fibrogenesis. The accumulation of fat sensitizes the liver to oxidative stress and triggers mechanisms like the activation of hepatic stellate cells (HSCs) and hepatocyte apoptosis or necrosis, central to the fibrotic process. While early-stage steatosis is reversible, chronic steatosis perpetuates a cycle of inflammation and fibrosis, creating a feedback loop that amplifies liver damage (Pafili K et al, 2021). Consequently, liver steatosis is not only a precursor but also a critical driver of fibrosis progression. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102. Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683. Description from EU-ToxRisk: Activation of stellate cells results in collagen accumulation and change in extracellular matrix composition in the liver causing fibrosis. (Landesmann, 2016; Koo et al 2016)

Steatosis is measured by lipidomics approaches (e.g. Yang and Han 2016) that measure lipid levels, or by histology.  Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, and other direct assessment techniques (Schaefer et al. 2016).

Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa. Life Stage: The life stage applicable to this key event is all life stages with a liver.  Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides. Sex: This key event applies to both males and females. Taxonomic: This key event appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

UBERON:0002107

UBERON liver

High Unspecific

High

All life stages

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. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102. Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2). https://doi.org/10.1016/j.molcel.2005.08.010   Koo, J. H., Lee, H. J., Kim, W., & Kim, S. G. (2016). Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2. Gastroenterology, 150(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039 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.   Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683. 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 2016-11-29T18:41:24

2026-02-11T05:41:52

Increase, Hepatocellular lipotoxicity

Cellular

AOPWiki 2026-02-10T04:40:07

2026-02-10T04:40:07

Increase, Oxidative Stress

Molecular

Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell.  As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al., 2009) and can be used as indicators of the presence of oxidative stress in the cell.  In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides on neighboring amino acids (Antelmann & Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).  ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O2. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017).   However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017).    Sources of ROS Production  Direct Sources: Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO2*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H2O2) and more O2 (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021).   Indirect Sources: An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008).  As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H2O2) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O2, and inorganic phosphate (Pi) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria has its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS is also produced through nicotinamide adenine dinucleotide phosphate oxidase (Nox) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).

Oxidative Stress: Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed  <ul> <li>Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683) </li> <li>Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential. </li> <li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green- ab138881.html). </li> <li>TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. </li> <li>8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015). </li> </ul>    Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:  <ul> <li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus Western blot for increased Nrf2 protein levels </li> <li>Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </li> <li>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014) </li> <li>OECD TG422D describes an ARE-Nrf2 Luciferase test method </li> </ul> In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation. <table border="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> ROS  Formation in the Mitochondria assay (Shaki et al., 2012)  </td> <td> “The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 µM) in respiration buffer containing (0.32 mM sucrose, 10mM Tris, 20 mM Mops, 50 µM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 µM) to mitochondria and was then incubated for 10 min.Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”    </td> <td> 0, 50,100 and 200 µM of Uranyl Acetate    </td> <td>  Long/ Easy High accuracy    </td> </tr> <tr> <td> Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012)    </td> <td> “GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as µg/mg protein.”  </td> <td> 0, 50,  100, or  200 µM  Uranyl Acetate  </td> <td>   </td> </tr> <tr> <td> H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria (Heyno et al., 2008)    </td> <td> “Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer  (see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.”   </td> <td> 0, 10, 30  µM Cd2+     2 µM antimycin A  </td> <td>   </td> </tr> <tr> <td> Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997)    </td> <td> “For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”  </td> <td>   </td> <td>           Strong/easy medium  </td> </tr> <tr> <td> DCFH-DA  Assay Detection of hydrogen peroxide production (Yuan et al.,  2016)  </td> <td> Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production.    </td> <td> 0-400  µM  </td> <td> Long/ Easy High accuracy  </td> </tr> <tr> <td> H2-DCF-DAAssay Detection of superoxide production (Thiebault etal., 2007)    </td> <td> This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.  </td> <td> 0–600  µM  </td> <td> Long/ Easy High accuracy  </td> </tr> <tr> <td> CM-H2DCFDA  Assay (Eruslanov  & Kusmartsev, 2009)  </td> <td> The dye (CM-H2DCFDA) diffuses into the cell and is cleaved by esterases, the thiol reactive chlormethyl group reacts with intracellular glutathione which can be detected using flow cytometry.  </td> <td>   </td> <td> Long/Easy/ High Accuracy  </td> </tr> </tbody> </table>   <table border="1"> <tbody> <tr> <td> Method of Measurement   </td> <td> References   </td> <td> Description   </td> <td colspan="2"> OECD-Approved Assay  </td> </tr> <tr> <td> Chemiluminescence   </td> <td> (Lu, C. et al., 2006;   Griendling, K. K., et al., 2016)  </td> <td> ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as luminol and lucigenin are commonly used to amplify the signal.   </td> <td colspan="2"> No    </td> </tr> <tr> <td> Spectrophotometry   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Direct or Spin Trapping-Based electron paramagnetic resonance (EPR) Spectroscopy   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> The unpaired electrons (free radicals) found in ROS can be detected with EPR and is known as electron paramagnetic resonance. A variety of spin traps can be used.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Nitroblue Tetrazolium Assay   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> The Nitroblue Tetrazolium assay is used to measure O2.− levels. O2.− reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Fluorescence analysis of DHE is used to measure O2.− levels.  O2.− is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Amplex Red Assay   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Dichlorodihydrofluorescein Diacetate (DCFH-DA)   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> An indirect fluorescence analysis to measure intracellular H2O2 levels.  H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> HyPer Probe   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Cytochrome c Reduction Assay   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> The cytochrome c reduction assay is used to measure O2.− levels. O O2.− is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Proton-electron double-resonance imaging (PEDRI)   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule.   </td> <td colspan="2"> No              </td> </tr> <tr> <td> Glutathione (GSH) depletion   </td> <td> (Biesemann, N. et al., 2018)   </td> <td> A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., <a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html" rel="noreferrer noopener" target="_blank">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>).    </td> <td colspan="2"> No  </td> </tr> <tr> <td> Thiobarbituric acid reactive substances (TBARS)   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit.    </td> <td colspan="2"> No  </td> </tr> <tr> <td> Protein oxidation (carbonylation)  </td> <td> (Azimzadeh et al., 2017; Azimzadeh et al., 2015; Ping et al., 2020)  </td> <td> Can be determined with ELISA or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.  </td> <td colspan="2"> No  </td> </tr> <tr> <td> Seahorse XFp Analyzer  </td> <td> Leung et al. 2018  </td> <td> The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR).  </td> <td> No  </td> </tr> </tbody> </table>   Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include:   <table border="1"> <tbody> <tr> <td> Method of Measurement   </td> <td> References   </td> <td> Description   </td> <td> OECD-Approved Assay  </td> </tr> <tr> <td> Immunohistochemistry   </td> <td> (Amsen, D., de Visser, K. E., and Town, T., 2009)  </td> <td> Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus    </td> <td> No  </td> </tr> <tr> <td> qPCR   </td> <td> (Forlenza et al., 2012)  </td> <td> qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)   </td> <td> No  </td> </tr> <tr> <td> Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis  </td> <td> (Jackson, A. F. et al., 2014)  </td> <td> Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway  </td> <td> No  </td> </tr> </tbody> </table>

Taxonomic applicability: Occurrence of oxidative stress is not species specific.   Life stage applicability: Occurrence of oxidative stress is not life stage specific.  Sex applicability: Occurrence of oxidative stress is not sex specific.  Evidence for perturbation by prototypic stressor: There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009).

High Mixed

High

All life stages

High High

Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, Journal Insect Science, Vol. 21/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jisesa/ieab080" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jisesa/ieab080</a>  Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3400</a>  Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, <a href="https://doi.org/10.1007/978-1-59745-447-6_5" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/978-1-59745-447-6_5</a>   Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/pr501141b</a>  Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1339332</a>  Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012  Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00520.2019." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00520.2019.</a>  Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a>    Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, Scientific Reports, Vol. 8/1, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41598-018-27614-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-018-27614-8</a>.   Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548  Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in molecular biology ,N.J.,  Vol. 594,  https://doi.org/10.1007/978-1-60761-411-1_4  Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a>   Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, https://doi.org/10.1007/978-1-61779-439-1_2   Forrester, S.J. et al. (2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, Physiological Reviews, Vol. 98/3, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00038.201" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00038.201</a>  Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814  Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, Current eye research, Vol. 31/1, Informa, London, <a href="https://doi.org/10.1080/02713680500477347.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a>   Griendling, K. K. et al. (2016), “Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association”, Circulation research, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/RES.0000000000000110" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/RES.0000000000000110</a>   Guo, C. et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, Neural regeneration research, Vol. 8/21, Publishing House of Neural Regeneration Research, China, <a href="https://doi.org/10.3969/j.issn.1673-5374.2013.21.009" rel="noreferrer noopener" target="_blank">https://doi.org/10.3969/j.issn.1673-5374.2013.21.009</a>  Hargreaves, M., and L. L. Spriet (2020), “Skeletal muscle energy metabolism during exercise.”, Nature Metabolism, Vol. 2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s42255-020-0251-4" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s42255-020-0251-4</a>  Hladik, D. and S. Tapio (2016), “Effects of ionizing radiation on the mammalian brain”, Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrrev.2016.08.003" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrrev.2016.08.003</a>  Itoh, K., J. Mimura and M. Yamamoto (2010), “Discovery of the negative regulator of Nrf2, Keap1: a historical overview”, Antioxidants & Redox Signaling, Vol. 13/11, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3222" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3222</a>   Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan.”, Toxicology and Applied Pharmacology, Vol. 274/11, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.taap.2013.10.019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.taap.2013.10.019</a>  Jacobsen, N.R. et al. (2008), “Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the FE1-MutaTM Mouse lung epithelial cells”, Environmental and Molecular Mutagenesis, Vol. 49/6, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1002/em.20406" rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/em.20406</a>  Karimi, N. et al. (2017), “Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats”, International Journal of Pharmaceutical Investigation, Vol. 7/3, Phcog Net, Bengaluru, <a href="https://doi.org/10.4103/jphi.JPHI_60_17.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.4103/jphi.JPHI_60_17. </a>  Leung, D.T.H., and Chu, S. (2018), “Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System” In: Murthi, P., Vaillancourt, C. (eds) Preeclampsia. Methods in Molecular Biology, vol 1710. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7498-6_22  Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, TrAC Trends in Analytical Chemistry, Vol. 25/10, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.trac.2006.07.007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.trac.2006.07.007</a>  Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, Antioxidants & redox signaling, Vol. 19/10, Mary Ann Liebert, Larchmont, <a href="https://doi.org/10.1089/ars.2012.4641" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2012.4641</a>  Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</a>  Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, Physiological Reviews, Vol. 88/4, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00031.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00031.2007</a>  Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, British Journal of Cancer, Vol. 122/2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41416-019-0651-y" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41416-019-0651-y</a>  Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, Acta Ophthalmologica, Vol. 96/4, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1111/aos.13526" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/aos.13526</a>  Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/gerona/glt057.</a>     Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, Life, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/life11111269" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/life11111269</a>  Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, International Journal of Biological Sciences, Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" rel="noreferrer noopener" target="_blank">https://doi.org/10.7150/ijbs.35460</a>   Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, Journal of applied physiology, Vol. 106/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.01278.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.01278.2007</a>.  Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, International journal of molecular medicine, Vol. 44/1, Spandidos Publishing Ltd., Athens, <a href="https://doi.org/10.3892/ijmm.2019.4188" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm.2019.4188</a>  AOPWiki 2017-05-30T13:58:17

2026-02-11T07:05:27

Increase, Cell injury/death

Cell injury/death

Cellular

Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased intracellular Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome. DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining (see explanation below). Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis. Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell (Fujikawa, 2015). Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process (Fujikawa, 2015; Malhi et al., 2010). Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA (Fujikawa, 2015; Malhi et al., 2010; Kaplowitz, 2002; Kroemer et al., 2009).

Necrosis: Lactate dehydrogenase (LDH) is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample (Chan et al., 2013).  The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes include XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light (Berridgeet al.,2005). Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm (Moore et al.,1998) Alamar Blue (resazurin) is a fluorescent dye. The oxidized blue non fluorescent Alamar blue is reduced to a pink fluorescent dye in the medium by cell activity (O'Brien et al., 2000) (12). Neutral red uptake, which is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto et al., 2008)(13). Moreover, quantification of ATP, signaling the presence of metabolically active cells, can be performed (CellTiter-Glo; Promega). ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega). Apoptosis: TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage. Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence (Li, Peng et al., 2004). Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis (Loo, 2002; Kubbies and Rabinovitch, 1983).  Acridine Orange/Ethidium Bromide staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis.

Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).

CL:0000255

CL eukaryotic cell

Not Specified Unspecific

Not Specified

All life stages

High High High High

<ul> <li>Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.</li> <li>Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.</li> <li>Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2, <a class="external free" href="http://www.medscape.com/viewarticle/433631" rel="nofollow" target="_blank">http://www.medscape.com/viewarticle/433631</a> (accessed on 20 January 2016).</li> <li>Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.</li> <li>Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.</li> <li>Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.</li> <li>Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.</li> <li>Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.</li> <li>Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.</li> <li>Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.</li> <li>Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.</li> <li>O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267(17): 5421-5426.</li> <li>Repetto G, del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols 3(7): 1125-1131.</li> </ul> AOPWiki 2016-11-29T18:41:22

2024-05-27T07:23:38

Increase, Kupffer cell activation

Cellular

Kupffer cells (KCs) are a specialized population of macrophages that reside in the liver; they were first described by Carl Wilhelm von Kupffer (1829–1902). <a href="#cite_note-1">[1]</a> KCs constitute 80%-90% of the tissue macrophages in the reticuloendothelial system and account for approximately 15% of the total liver cell population <a href="#cite_note-2">[2]</a> They play an important role in normal physiology and homeostasis as well as participating in the acute and chronic responses of the liver to toxic compounds. Activation of KCs results in the release of an array of inflammatory mediators, growth factors, and reactive oxygen species. This activation appears to modulate acute hepatocyte injury as well as chronic liver responses including hepatic cancer. Understanding the role KCs play in these diverse responses is key to understanding mechanisms of liver injury.<a href="#cite_note-Roberts2007-3">[3]</a> Besides the release of inflammatory mediators including cytokines, chemokines, lysosomal and proteolytic enzymes KCs are a main source of TGF-β1 (transforming growth factor-beta 1, the most potent profibrogenic cytokine). In addition latent TGF-β1 can be activated by KC-secreted matrix metalloproteinase 9 (MMP-9). <a href="#cite_note-4">[4]</a> <a href="#cite_note-Luckey_2001-5">[5]</a> through the release of biologically active substances that promote the pathogenic process. Activated KCs also release ROS like superoxide generated by NOX (NADPH oxidase), thus contributing to oxidative stress. Oxidative stress also activates a variety of transcription factors like NF-κB, PPAR-γ leading to an increased gene expression for the production of growth factors, inflammatory cytokines and chemokines. KCs express TNF-α (Tumor Necrosis Factor-alpha), IL-1 (Interleukin-1) and MCP-1 (monocyte-chemoattractant protein-1), all being mitogens and chemoattractants for hepatic stellate ceells (HSCs) and induce the expression of PDGF receptors on HSCs which enhances cell proliferation. Expressed TNF-α, TRAIL (TNF-related apoptosis-inducing ligand), and FasL (Fas Ligand) are not only pro-inflammatory active but also capable of inducing death receptor-mediated apoptosis in hepatocytes<a href="#cite_note-6">[6]</a> <a href="#cite_note-7">[7]</a><a href="#cite_note-Roberts2007-3">[3]</a> Under conditions of oxidative stress macrophages are further activated which leads to a more enhanced inflammatory response that again further activates KCs though cytokines (Interferon gamma (IFNγ), granulocyte macrophage colony-stimulating factor (GM-CSF), TNF-α), bacterial lipopolysaccharides, extracellular matrix proteins, and other chemical mediators. <a href="#cite_note-8">[8]</a> <a href="#cite_note-9">[9]</a> Besides KCs, the resident hepatic macrophages, infiltrating bone marrow-derived macrophages, originating from circulating monocytes are recruited to the injured liver via chemokine signals. KCs appear essential for sensing tissue injury and initiating inflammatory responses, while infiltrating Ly-6C+ monocyte-derived macrophages are linked to chronic inflammation and fibrogenesis. The profibrotic functions of KCs (HSC activation via paracrine mechanisms) during chronic hepatic injury remain functionally relevant, even if the infiltration of additional inflammatory monocytes is blocked via pharmacological inhibition of the chemokine CCL2 <a href="#cite_note-10">[10]</a> <a href="#cite_note-11">[11]</a> KC activation and macrophage recruitment are two separate events and both are necessary for fibrogenesis, but as they occur in parallel, they can be summarised as one KE. Probably there is a threshold of KC activation and release above which liver damage is induced. Pre-treatment with gadolinium chloride (GdCl), which inhibits KC function, reduced both hepatocyte and sinusoidal epithelial cell injury, as well as decreased the numbers of macrophages appearing in hepatic lesions and inhibited TGF-β1 mRNA expression in macrophages. Experimental inhibition of KC function or depletion of KCs appeared to protect against chemical-induced liver injury.<a href="#cite_note-12">[12]</a>

Kupffer cell activation can be measured by means of expressed cytokines, e.g. tissue levels of TNF-a <a href="#cite_note-13">[13]</a>, IL-6 expression, measured by immunoassays or Elisa (offered by various companies), soluble CD163 <a href="#cite_note-14">[14]</a> <a href="#cite_note-15">[15]</a> or increase in expression of Kupffer cell marker genes such as Lyz, Gzmb, and Il1b, (Genome U34A Array, Affymetrix); <a href="#cite_note-16">[16]</a>

Human: <a href="#cite_note-17">[17]</a><a href="#cite_note-18">[18]</a><a href="#cite_note-19">[19]</a> Rat: <a href="#cite_note-Luckey_2001-5">[5]</a> Mouse: <a href="#cite_note-20">[20]</a>

UBERON:0002107

UBERON liver

CL:0000091

CL Kupffer cell

High High High High

<ol> <li><a href="#cite_ref-1">↑</a> Haubrich, W.S. (2004), Kupffer of Kupffer cells, Gastroenterology, vol. 127, no. 1, p. 16.</li> <li><a href="#cite_ref-2">↑</a> Bouwens, L. et al. (1986), Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver, Hepatology, vol. 6, no. 6, pp. 718-722.</li> <li>↑ <a href="#cite_ref-Roberts2007_3-0">3.0</a> <a href="#cite_ref-Roberts2007_3-1">3.1</a> Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15.</li> <li><a href="#cite_ref-4">↑</a> Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.</li> <li>↑ <a href="#cite_ref-Luckey_2001_5-0">5.0</a> <a href="#cite_ref-Luckey_2001_5-1">5.1</a> Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</li> <li><a href="#cite_ref-6">↑</a> Guo, J. and S.L. Friedman (2007), Hepatic Fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</li> <li><a href="#cite_ref-7">↑</a> Friedman, S.L. (2002), Hepatic Fibrosis-Role of Hepatic Stellate Cell Activation, MedGenMed, vol. 4, no. 3, pp. 27.</li> <li><a href="#cite_ref-8">↑</a> Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer Cells in the Pathogenesis of Liver Disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.</li> <li><a href="#cite_ref-9">↑</a> Kershenobich Stalnikowitz, D. and A.B. Weisssbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</li> <li><a href="#cite_ref-10">↑</a> Baeck, C. et al. (2012), Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury, Gut, vol. 61, no. 3, pp.416–426.</li> <li><a href="#cite_ref-11">↑</a> Tacke, F. and H.W. Zimmermann (2014), Macrophage heterogeneity in liver injury and fibrosis, J Hepatol, vol. 60, no. 5, pp. 1090-1096.</li> <li><a href="#cite_ref-12">↑</a> Ide, M. et al. (2005), Effects of gadolinium chloride (GdCl(3)) on the appearance of macrophage populations and fibrogenesis in thioacetamide-induced rat hepatic lesions, J. Comp. Path, vol. 133, no. 2-3, pp. 92–102.</li> <li><a href="#cite_ref-13">↑</a> Vajdova, K. et al. (2004), Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury, Liver Transpl, vol. 10, no. 4, pp. 520–528.</li> <li><a href="#cite_ref-14">↑</a> Grønbaek, H. et al. (2012), Soluble CD163, a marker of Kupffer cell activation, is related to portal hypertension in patients with liver cirrhosis, Aliment Pharmacol Ther, vol 36, no. 2, pp. 173-180.</li> <li><a href="#cite_ref-15">↑</a> Møller, H.J. (2012), Soluble CD163.Scand J Clin Lab Invest, vol. 72, no. 1, pp. 1-13.</li> <li><a href="#cite_ref-16">↑</a> Takahara, T et al. (2006), Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis, World J Gastroenterol, vol. 12, no. 40, pp. 6473-6499.</li> <li><a href="#cite_ref-17">↑</a> Su, G.L. et al. (2002), Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14, Am J Physiol Gastrointest Liver Physiol, vol. 283, no. 3, pp. G640-645.</li> <li><a href="#cite_ref-18">↑</a> Kegel, V. et al. (2015), Subtoxic concentrations of hepatotoxic drugs lead to Kupffer cell activation in a human in vitro liver model: an approach to study DILI, Mediators Inflamm, 2015:640631, <a class="external free" href="http://doi.org/10.1155/2015/640631" rel="nofollow" target="_blank">http://doi.org/10.1155/2015/640631</a>.</li> <li><a href="#cite_ref-19">↑</a> Boltjes, A. et al. (2014), The role of Kupffer cells in hepatitis B and hepatitis C virus infections, J Hepatol, vol. 61, no. 3, pp. 660-671.</li> <li><a href="#cite_ref-20">↑</a> Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.</li> </ol> AOPWiki 2016-11-29T18:41:22

2026-02-11T05:16:36

Increase, Steatohepatitis

Organ

Steatohepatitis is characterized by hepatic steatosis accompanied by hepatocellular injury and lobular inflammation. It represents a pathological progression beyond simple steatosis and is a defining feature of metabolic dysfunction–associated steatohepatitis (MASH). Histologically, steatohepatitis includes: <ul> <li> Macrovesicular steatosis </li> <li> Hepatocyte ballooning degeneration </li> <li> Lobular inflammatory cell infiltration </li> <li> Mallory–Denk bodies (in some cases) </li> <li> Variable degrees of perisinusoidal fibrosis </li> </ul>

<h3>1. Histopathology (Primary Method)</h3> <ul> <li> Hematoxylin and eosin (H&E) staining </li> <li> NAFLD Activity Score (NAS) </li> <li> Steatosis–Activity–Fibrosis (SAF) scoring system </li> <li> Assessment of: <ul> <li> Steatosis grade </li> <li> Ballooning degeneration </li> <li> Lobular inflammation </li> </ul> </li> </ul> Histological scoring systems are considered the gold standard for detection. <h3>2. Serum Biomarkers</h3> <ul> <li> Elevated ALT (alanine aminotransferase) </li> <li> Elevated AST (aspartate aminotransferase) </li> <li> Inflammatory cytokines (TNF-α, IL-6) </li> </ul> Biochemical markers provide supportive but not definitive evidence. <h3>3. Molecular Markers</h3> <ul> <li> Increased expression of inflammatory genes (e.g., TNF-α, MCP-1) </li> <li> Markers of hepatocyte injury (e.g., cytokeratin-18 fragments) </li> </ul> <h3>4. Imaging (Clinical Context)</h3> <ul> <li> MRI-PDFF (steatosis quantification) </li> <li> Elastography (for associated fibrosis) </li> </ul> However, histology remains required for definitive diagnosis of steatohepatitis.

Steatohepatitis is a well-defined pathological entity in humans and is reproducibly induced in rodent models of metabolic dysfunction and lipotoxic stress. The KE is most applicable to: <ul> <li> Mammalian species with comparable hepatic architecture </li> <li> Conditions involving chronic metabolic stress </li> <li> Adult or metabolically mature organisms </li> </ul> The weight of evidence for this KE is strong due to consistent clinical and experimental characterization.

UBERON:0002107

UBERON liver

High Unspecific

High

All life stages

High AOPWiki 2017-11-13T12:47:00

2026-02-24T09:13:44

Increase, Transforming growth factor-beta signaling

Activation of TGF-β signaling

Molecular

AOPWiki 2017-02-15T02:45:16

2026-02-11T05:39:19

Increase, Hepatic stellate cell activation

Increase, HSC activation

Cellular

Stellate cell activation means a transdifferentiation from a quiescent vitamin A–storing cell to a proliferative and contractile myofibroblast. Multiple cells and cytokines play a part in the regulation of hepatic stellate cell (HSC) activation that consists of discrete phenotype responses, mainly proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis, and retinoid loss. HSCs undergo activation through a two-phase process. The first step, the initiation phase, is triggered by injured hepatocytes, reactive oxygen speecies (ROS) and paracrine stimulation from neighbouring cell types (Kupffer cells (KCs), Liver sinusoidal endothelial cells (LSECs), and platelets) and make HSCs sensitized to activation by up-regulating various receptors. The perpetuation phase refers to the maintenance of HSC activation, which is a dynamic process including the secretion of autocrine and paracrine growth factors (such as TGF-β1), chemokines, and the up-regulation of collagen synthesis (mainly type I collagen). In response to growth factors (including Platelet-derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF)) HSCs proliferate. Increased contractility (Endothelin-1 and NO are the key opposing counter-regulators that control HSC contractility, in addition to angiotensinogen II, and others) leads to increased portal resistance. Driven by chemoattractants their accumulation in areas of injury is enhanced. TGF-β1 synthesis promotes activation of neighbouring quiescent hepatic stellate cells, whereas the release of HGF (hepatocyte growth factor) stimulates regeneration of adjacent hepatocytes. The release of chemoattractants (monocyte chemoattractant protein-1(MCP-1) and colony-stimulating factors (CSFs)) amplifies inflammation (Lee and Friedman; 2011; Friedman, 2010; 2008; 2000; Bataller and Brenner, 2005; ↑ Lotersztain et al., 2005; Poli, 2000). Activated HSCs (myofibroblasts) are the primary collagen producing cell, the key cellular mediators of fibrosis and a nexus for converging inflammatory pathways leading to fibrosis. Experimental inhibition of stellate cell activation prevents fibrosis (Li, Jing-Ting et al.,2008; George et al. (1999).

Alpha-smooth muscle actin (α-SMA) is a well-known marker of hepatic stellate cells activation. Anti-alpha smooth muscle Actin [1A4] monoclonal antibody reacts with the alpha smooth muscle isoform of actin. Gene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected and as marker for culture-activated HSCs and plays a role in HSC migration (Morini et al., 2005; Mannaerts et al., 2013).    <pre>  </pre>

Human: Friedman, 2008 Rat: George et al.,1999 Mouse: Chang et al., 2014 Pig: Costa et al., 2001

UBERON:0002107

UBERON liver

CL:0000632

CL hepatic stellate cell

Not Specified Unspecific

Not Specified

All life stages

High High High High High

<ul> <li>Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract Res Clin Gastroenterol, vol. 25, no. 2, pp. 195-206.</li> <li>Friedman, S.L (2010), Evolving challenges in hepatic fibrosis, Nat. Rev. Gastroenterol. Hepatol, vol. 7, no. 8, pp. 425–436.</li> <li>Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.</li> <li>Friedman, S.L (2000), Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury, J. Biol. Chem, vol. 275, no. 4, pp. 2247-2250.</li> <li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li> <li>Lotersztain, S. et al. (2005), Hepatic fibrosis: molecular mechanisms and drug targets, Annu. Rev. Pharmacol. Toxicol, vol. 45, pp. 605–628.</li> <li>Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98.</li> <li>Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.</li> <li>George, J. et al. (1999), In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: a potential new therapy for hepatic fibrosis. Proc Natl Acad Sci, vol. 96, no. 22, pp. 12719-12724.</li> <li>Morini, S. et al. (2005), GFAP expression in the liver as an early marker of stellate cells activation, Ital J Anat Embryol, vol. 110, no. 4, pp. 193-207.</li> <li>Mannaerts, I. et al. (2013), Gene expression profiling of early hepatic stellate cell activation reveals a role for Igfbp3 in cell migration, PLoS One, vol. 8, no.12, e84071.</li> <li>Chang et al., 2014, Isolation and culture of hepatic stellate cells from mouse liver. Acta Biochim Biophys Sin (Shanghai).;46(4):291-8.</li> <li>Costa et al., 2001, Early activation of hepatic stellate cells and perisinusoidal extracellular matrix changes during ex vivo pig liver perfusion. J Submicrosc Cytol Pathol.;33(3):231-40.</li> </ul> AOPWiki 2016-11-29T18:41:23

2026-02-11T07:04:19

Increase, Collagen accumulation

Tissue

Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. It is also abundant in corneas, cartilage, bones, blood vessels, the gut, intervertebral discs, and the dentin in teeth. In muscle tissue, it serves as a major component of the endomysium. Collagen is the main structural protein in the extracellular space in the various connective tissues, making up from 25% to 35% of the whole-body protein content. In normal tissues, collagen provides strength, integrity, and structure. When tissues are disrupted following injury, collagen is needed to repair the defect. If too much collagen is deposited, normal anatomical structure is lost, function is compromised, and fibrosis results. The fibroblast is the most common collagen producing cell. Collagen-producing cells may also arise from the process of transition of differentiated epithelial cells into mesenchymal cells. This has been observed e.g. during renal fibrosis (transformation of tubular epithelial cells into fibroblasts) and in liver injury (transdifferentiation of hepatocytes and cholangiocytes into fibroblasts) (Henderson and Iredale, 2007). There are close to 20 different types of collagen found with the predominant form being type I collagen. This fibrillar form of collagen represents over 90 percent of our total collagen and is composed of three very long protein chains which are wrapped around each other to form a triple helical structure called a collagen monomer. Collagen is produced initially as a larger precursor molecule called procollagen. As the procollagen is secreted from the cell, procollagen proteinases remove the extension peptides from the ends of the molecule. The processed molecule is referred to as collagen and is involved in fiber formation. In the extracellular spaces the triple helical collagen molecules line up and begin to form fibrils and then fibers. Formation of stable crosslinks within and between the molecules is promoted by the enzyme lysyl oxidase and gives the collagen fibers tremendous strength (Diegelmann,2001). The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Disturbance of this balance leads to changes in the amount and composition of collagen. Changes in the composition of the extracellular matrix initiate positive feedback pathways that increase collagen production. Normally, collagen in connective tissues has a slow turn over; degradating enzymes are collagenases, belonging to the family of matrix metalloproteinases. Other cells that can synthesize and release collagenase are macrophages, neutrophils, osteoclasts, and tumor cells (Di Lullo et al., 2002; Kivirikko and Risteli, 1976; Miller and Gay, 1987; Prockop and Kivirikko, 1995).

Determination of the amount of collagen produced in vitro can be done in a variety of ways ranging from simple colorimetric assays to elaborate chromatographic procedures using radioactive and non-radioactive material. What most of these procedures have in common is the need to destroy the cell layer to obtain solubilized collagen from the pericellular matrix. Rishikof et al. describe several methods to assess the in vitro production of type I collagen: Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides, the measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction, and methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay (Rishikof et al., 2005).  Histological staining with stains such as Masson Trichrome, Picro-sirius red are used to identify the tissue/cellular distribution of collagen, which can be quantified using morphometric analysis both in vivo and in vitro. The assays are routinely used and are quantitative. Sircol Collagen Assay for collagen quantification: The Serius dye has been used for many decades to detect collagen in histology samples. The Serius Red F3BA selectively binds to collagen and the signal can be read at 540 nm (Chen and Raghunath, 2009; Nikota et al., 2017). Hydroxyproline assay: Hydroxyproline is a non-proteinogenic amino acid formed by the prolyl-4-hydroxylase. Hydroxyproline is only found in collagen and thus, it serves as a direct measure of the amount of collagen present in cells or tissues. Colorimetric methods are readily available and have been extensively used to quantify collagen using this assay (Chen and Raghunath, 2009; Nikota et al., 2017). Ex vivo precision cut tissue slices Precision cut tissue slices mimic the whole organ response and allow histological assessment, an endpoint of interest in regulatory decision making. While this technique uses animals, the number of animals required to conduct a dose-response study can be reduced to 1/4th of what will be used in whole animal exposure studies (Rahman et al., 2020).    <pre>  </pre>

Humans: Bataller and  Brenner, 2005; Decaris et al., 2015.   Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013. Rats: Hamdy and El-Demerdash, 2012; Li et al., 2012; Luckey and Petersen, 2001; Natajaran et al., 2006.

UBERON:0002384

UBERON connective tissue

Not Specified Unspecific

Not Specified

All life stages

High High High

<ol> <li> Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005 Feb;115(2):209-18. doi: 10.1172/JCI24282.  </li> <li> Chen CZ, Raghunath M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art. Fibrogenesis Tissue Repair. 2009 Dec 15;2:7. doi: 10.1186/1755-1536-2-7.  </li> <li> Dalton SR, Lee SM, King RN, Nanji AA, Kharbanda KK, Casey CA, McVicker BL. Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice. Biochem Pharmacol. 2009 Apr 1;77(7):1283-90. doi: 10.1016/j.bcp.2008.12.023.  </li> <li> Decaris ML, Emson CL, Li K, Gatmaitan M, Luo F, Cattin J, Nakamura C, Holmes WE, Angel TE, Peters MG, Turner SM, Hellerstein MK. Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease. PLoS One. 2015 Apr 24;10(4):e0123311. doi: 10.1371/journal.pone.0123311. </li> <li> Di Lullo GA, Sweeney SM, Korkko J, Ala-Kokko L, San Antonio JD. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem. 2002 Feb 8;277(6):4223-31. doi: 10.1074/jbc.M110709200. </li> <li> Diegelmann R. Collagen Metabolism. Wounds. 2001;13:177-82. Available at www.medscape.com/viewarticle/423231 (accessed on 20 January 2016). </li> <li> Hamdy N, El-Demerdash E. New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage. Toxicol Appl Pharmacol. 2012 Jun 15;261(3):292-9. doi: 10.1016/j.taap.2012.04.012.  </li> <li> Henderson NC, Iredale JP. Liver fibrosis: cellular mechanisms of progression and resolution. Clin Sci (Lond). 2007 Mar;112(5):265-80. doi: 10.1042/CS20060242. </li> <li> Kivirikko KI, Risteli L. Biosynthesis of collagen and its alterations in pathological states. Med Biol. 1976 Jun;54(3):159-86. </li> <li> Leung TM, Tipoe GL, Liong EC, Lau TY, Fung ML, Nanji AA. Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis. Int J Exp Pathol. 2008 Aug;89(4):241-50. doi: 10.1111/j.1365-2613.2008.00590.x.  </li> <li> Li L, Hu Z, Li W, Hu M, Ran J, Chen P, Sun Q. Establishment of a standardized liver fibrosis model with different pathological stages in rats. Gastroenterol Res Pract. 2012;2012:560345. doi: 10.1155/2012/560345.  </li> <li> Luckey SW, Petersen DR. Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats. Exp Mol Pathol. 2001 Dec;71(3):226-40. doi: 10.1006/exmp.2001.2399. </li> <li> Miller EJ, Gay S. The collagens: an overview and update. Methods Enzymol. 1987;144:3-41. doi: 10.1016/0076-6879(87)44170-0.  </li> <li> Nan YM, Kong LB, Ren WG, Wang RQ, Du JH, Li WC, Zhao SX, Zhang YG, Wu WJ, Di HL, Li Y, Yu J. Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice. Lipids Health Dis. 2013 Feb 6;12:11. doi: 10.1186/1476-511X-12-11. </li> <li> Natarajan SK, Thomas S, Ramamoorthy P, Basivireddy J, Pulimood AB, Ramachandran A, Balasubramanian KA. Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models. J Gastroenterol Hepatol. 2006 Jun;21(6):947-57. doi: 10.1111/j.1440-1746.2006.04231.x. </li> <li> Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0.  </li> <li> Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem. 1995;64:403-34. doi: 10.1146/annurev.bi.64.070195.002155.  </li> <li> Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272. </li> <li> Rishikof DC, Kuang PP, Subramanian M, Goldstein RH. Methods for measuring type I collagen synthesis in vitro. Methods Mol Med. 2005;117:129-40. doi: 10.1385/1-59259-940-0:129.  </li> </ol> AOPWiki 2016-11-29T18:41:22

2026-02-11T06:58:07

Increase, Liver fibrosis

Organ

Liver fibrosis results from perpetuation of the normal wound healing response, as a result of repeated cycles of hepatocyte injury and repair and is a dynamic process, characterised by an excessive deposition of ECM (extracellular matrix) proteins including glycoproteins, collagens, and proteoglycans. It is usually secondary to hepatic injury and inflammation, and progresses at different rates depending on the aetiology of liver disease and is also influenced by environmental and genetic factors. If fibrosis continues, it disrupts the normal architecture of the liver, altering the normal function of the organ and ultimately leading to liver damage. Cirrhosis represents the final stage of fibrosis. It is characterised by fibrous septa which divide the parenchyma into regenerative nodules which leads to vascular modifications and portal hypertension with its complications of variceal bleeding, hepatic encephalopathy, ascites, and hepatorenal syndrome. In addition, this condition is largely associated with hepatocellular carcinoma with a further increase in the relative mortality rate (Bataller and Brenner, 2005; Merck Manual,2015) Liver fibrosis is an important health issue with clear regulatory relevance. The burden of disease attributable to liver fibrosis is quite high; progressive hepatic fibrosis, ultimately leading to cirrhosis, is a significant contributor to global health burden (Lim and Kim, 2008). In the European Union, 0.1 % of the population is affected by cirrhosis, the most advanced stage of liver fibrosis with full architectural disturbances (Blachier et al., 2013). Besides the epidemiological relevance, liver fibrosis also imposes a considerable economic burden on society. Indeed, the only curative therapy for chronic liver failure is liver transplantation. More than 5.500 orthotopic liver transplantations are currently performed in Europe on a yearly basis, costing up to €100.000 the first year and €10.000 yearly thereafter (Van Agthoven et al., 2001).

Liver biopsy is an important part of the evaluation of patients with a variety of liver diseases. Besides establishing the diagnosis, the biopsy is often used to assess the severity of the disease. Until recently it has been assumed that fibrosis is an irreversible process, so most grading and staging systems have relatively few stages and are not very sensitive for describing changes in fibrosis. In all systems, the stages are determined by both the quantity and location of the fibrosis, with the formation of septa and nodules as major factors in the transition from one stage to the next. The absolute amount of fibrous tissue is variable within each stage, and there is considerable overlap between stages. Commonly used systems are the Knodell score with 4 stages - no fibrosis (score 0) to fibrous portal expansion (score 2) to bridging fibrosis (score 3) and Cirrhosis (score 4) – and the more sensitive Ishak fibrosis score with six stages - from no fibrosis (stage 0) over increasing fibrous expansion on portal areas (stages 1-2), bridging fibrosis (stages 3-4), and nodules (stage 5) to cirrhosis (stage 6) (Goodman, 2007). Liver biopsy is an invasive test with many possible complications and the potential for sampling error. Noninvasive tests become increasingly precise in identifying the amount of liver fibrosis through computer-assisted image analysis. Standard liver tests are of limited value in assessing the degree of fibrosis. Direct serologic markers of fibrosis include those associated with matrix deposition — e.g.procollagen type III amino-terminal peptide (P3NP), type I and IV collagens, laminin, hyaluronic acid, and chondrex. P3NP is the most widely studied marker of hepatic fibrosis. Other direct markers of fibrosis are those associated with matrix degradation, ie, matrix metalloproteinases 2 and 3 (MMP-2, MMP- 3) and tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1, TIMP-2).These tests are not commercially available, and the components are not readily available in most clinical laboratories. Some indirect markers that combine several parameters are available but not very reliable. Conventional imaging studies (ultrasonography and computed tomography) are not sensitive for fibrosis. Hepatic elastography, a method for estimating liver stiffness, is a recent development in the noninvasive measurement of hepatic fibrosis. Currently, elastography can be accomplished by ultrasound or magnetic resonance. Liver biopsy is still needed if laboratory testing and imaging studies are inconclusive (Carey, 2010; Germani et al., 2011) .

Human: Bataller and Brenner, 2005;Merck Manual, 2015; Blachier et al., 2013. Rat, mouse: Liedtke et al., 2013

UBERON:0002107

UBERON liver

Not Specified Unspecific

Not Specified

All life stages

High High High

<ul> <li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li> <li>Merck Manual available at: <a class="external free" href="http://www.merckmanuals.com/professional/hepatic_and_biliary_disorders/fibrosis_and_cirrhosis/hepatic_fibrosis.html,(accessed" rel="nofollow" target="_blank">http://www.merckmanuals.com/professional/hepatic_and_biliary_disorders/fibrosis_and_cirrhosis/hepatic_fibrosis.html,(accessed</a> 10 February 2015).</li> <li>Lim, Y. and W. Kim (2008), The global impact of hepatic fibrosis and end-stage liver disease, Clin Liver Dis, vol. 12, no. 4, pp. 733-746.</li> <li>Blachier, M. et al. (2013), The burden of liver disease in Europe: a review of available epidemiological data, J Hepatol, vol. 58, no. 3, pp. 593-608.</li> <li>Van Agthoven, M. et al. (2001), A comparison of the costs and effects of liver transplantation for acute and for chronic liver failure. Transpl Int, vol. 14, no. 2, pp. 87-94.</li> <li>Goodman, Z.D. (2007), Grading and staging systems for inflammation and fibrosis in chronic liver diseases, Journal of Hepatology, vol. 47, no. 4, pp. 598-607.</li> <li>Carey, E. (2010), Noninvasive tests for liver disease, fibrosis, and cirrhosis: Is liver biopsy obsolete? Cleveland Clinic Journal of Medicine, vol. 77, no. 8, pp. 519-527.</li> <li>Germani, G. et al. (2011), Assessment of Fibrosis and Cirrhosis in Liver Biopsies, Semin Liver Dis, vol. 31, no. 1, pp. 82-90. available at <a class="external free" href="http://www.medscape.com/viewarticle/743946_2,(accessed" rel="nofollow" target="_blank">http://www.medscape.com/viewarticle/743946_2,(accessed</a> 10 February 2015).</li> <li>Liedtke, C. et al. (2013), Experimental liver fibrosis research: update on animal models, legal issues and translational aspects, Fibrogenesis Tissue Repair, vol. 6, no. 1, p. 19.</li> </ul> AOPWiki 2016-11-29T18:41:24

2026-02-11T05:35:21

Increase, Regenerative nodule formation

Tissue

UBERON:0002107

UBERON liver

AOPWiki 2026-02-10T04:47:33

2026-02-10T06:47:33

Increase, Cirrhosis

Organ

Cirrhosis represents the end-stage consequence of chronic liver injury and sustained fibrogenesis. It is characterized by: <ul> <li> Extensive deposition of extracellular matrix (primarily type I collagen) </li> <li> Bridging fibrosis connecting portal and central regions </li> <li> Formation of regenerative nodules </li> <li> Distortion of normal hepatic architecture </li> <li> Altered vascular structure and portal hypertension </li> </ul>

<h3>1. Histopathology (Gold Standard)</h3> <ul> <li> Masson's trichrome staining </li> <li> Sirius Red staining for collagen deposition </li> <li> Assessment of: <ul> <li> Bridging fibrosis </li> <li> Nodular regeneration </li> <li> Architectural distortion </li> </ul> </li> </ul> Common scoring systems: <ul> <li> METAVIR (F4 stage indicates cirrhosis) </li> <li> Ishak fibrosis score </li> <li> SAF scoring system </li> </ul> <h3>2. Imaging (Clinical Context)</h3> <ul> <li> Transient elastography (FibroScan) </li> <li> Magnetic resonance elastography </li> <li> Ultrasound imaging (nodular surface, splenomegaly) </li> </ul> Imaging provides non-invasive assessment but histology confirms diagnosis. <h3>3. Serum Biomarkers (Supportive)</h3> <ul> <li> Decreased albumin </li> <li> Elevated bilirubin </li> <li> Prolonged prothrombin time </li> <li> Fibrosis biomarkers (e.g., hyaluronic acid, procollagen peptides) </li> </ul> These support functional impairment but do not independently confirm cirrhosis.

Cirrhosis is a clinically recognized end-stage liver disease in humans and is reproducible in mammalian models of chronic hepatic injury. The mechanisms of fibrosis progression and architectural remodeling are highly conserved in mammals. This KE is most applicable under: <ul> <li> Chronic exposure conditions </li> <li> Sustained inflammatory and fibrogenic signaling </li> <li> Adult or metabolically mature organisms </li> </ul> The biological plausibility is strong due to well-established fibrogenic pathways (e.g., TGF-β–mediated stellate cell activation) and consistent cross-species pathology.

UBERON:0002107

UBERON liver

Not Specified Unspecific

Not Specified

Adult

Not Specified Not Specified Not Specified Not Specified AOPWiki 2026-02-10T04:48:48

2026-02-11T07:34:30

<upstream-id>2c1e29eb-34f8-4319-98f5-1364164a6680</upstream-id> <downstream-id>6a7a4994-3e84-4a8b-aec9-5883f2ecb444</downstream-id>

AOPWiki 2026-02-24T08:32:30

2026-02-24T08:32:30

<upstream-id>6a7a4994-3e84-4a8b-aec9-5883f2ecb444</upstream-id> <downstream-id>227e1b35-57bc-4c90-a3f4-7b6a27edabeb</downstream-id>

AOPWiki 2026-02-24T08:32:45

2026-02-24T08:32:45

<upstream-id>227e1b35-57bc-4c90-a3f4-7b6a27edabeb</upstream-id> <downstream-id>f83473dd-0a78-4f0d-aaf5-b1f3db16cd40</downstream-id>

AOPWiki 2026-02-12T07:41:40

2026-02-12T07:41:40

<upstream-id>f83473dd-0a78-4f0d-aaf5-b1f3db16cd40</upstream-id> <downstream-id>37fce6ae-eb76-4936-9a7f-e486cbf49601</downstream-id>

AOPWiki 2026-02-11T05:41:20

2026-02-11T05:41:20

<upstream-id>37fce6ae-eb76-4936-9a7f-e486cbf49601</upstream-id> <downstream-id>86da0329-f8db-48b3-bdb6-d7f1ea46abe2</downstream-id>

AOPWiki 2026-02-10T08:59:43

2026-02-10T08:59:43

<upstream-id>86da0329-f8db-48b3-bdb6-d7f1ea46abe2</upstream-id> <downstream-id>443aff71-7fdd-4d68-9973-37a3a9a4f9a1</downstream-id>

AOPWiki 2026-02-24T08:47:41

2026-02-24T08:47:41

<upstream-id>443aff71-7fdd-4d68-9973-37a3a9a4f9a1</upstream-id> <downstream-id>ee9118a3-6ea8-4f74-982c-75680fa3ba62</downstream-id> Oxidative stress (OS) as a concept in redox biology and medicine has been formulated in 1985 (Sies, 2015). OS is intimately linked to cellular energy balance and comes from the imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS) or from a decay of the antioxidant protective ability. OS is characterized by the reduced capacity of endogenous systems to fight against the oxidative attack directed towards target biomolecules (Wang and Michaelis, 2010; Pisoschi and Pop, 2015).  Glutathione, the most important redox buffer in cells (antioxidant), cycles between reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), and serves as a vital sink for control of ROS levels in cells (Reynolds et al., 2007).  Several case-control studies have reported the link between lower concentrations of GSH, higher levels of GSSG and the development of diseases (Rossignol and Frye, 2014). OS can cause cellular damage and subsequent cell death because the ROS oxidize vital cellular components such as lipids, proteins, and nucleic acids (Gilgun-Sherki, Melamed and Offen, 2001; Wang and Michaelis, 2010). The central nervous system is especially vulnerable to free radical damage since it has a high oxygen consumption rate, an abundant lipid content and reduced levels of antioxidant enzymes (Coyle and Puttfarcken, 1993; Markesbery, 1997). It has been show that the developing brain is particularly vulnerable to neurotoxicants and OS due to differentiation processes, changes in morphology, lack of physiological barriers and less intrinsic capacity to cope with cellular stress (Grandjean and Landrigan, 2014; Sandström et al., 2017). However, it has to be noted that neural stem cells distinguish themeselves from post-mitotic neural cells by their lower ROS levels and higher expression of the key antioxidant enzymes glutathione peroxidase. This increased "vigilance" of antioxidant mechanisms might represent an innate characteristic of NSCs, which not only defines their cell fate, but also helps them to encounter oxidative stress (Madhavan et al., 2006). OS has been linked to brain aging, neurodegenerative diseases, and other related adverse conditions.  There is evidence that free radicals play a role in cerebral ischemia-reperfusion, head injury, Parkinson’s disease, amyotrophic lateral sclerosis, Down’s syndrome, and Alzheimer’s disease due to cellular damage (Markesbery, 1997; Gilgun-Sherki, Melamed and Offen, 2001; Wang and Michaelis, 2010). OS has also been linked to neurodevelopmental diseases and deficits like autism spectrum disorder and postnatal motor coordination deficits (Wells et al., 2009; Rossignol and Frye, 2014; Bhandari and Kuhad, 2015).

A noteworthy insight, early on, was the perception that oxidation-reduction (redox) reactions in living cells are utilized in fundamental processes of redox regulation, collectively termed ‘redox signaling’ and ‘redox control’ (Sies, 2015). Free radical-induced damage in OS has been confirmed as a contributor to the pathogenesis and patho-physiology of many chronic diseases, such as Alzheimer, atherosclerosis, Parkinson, but also in traumatic brain injury, sepsis, stroke, myocardial infraction, inflammatory diseases, cataracts and cancer (Bar-Or et al., 2015; Pisoschi and Pop, 2015). It has been assessed that oxidative stress is correlated with over 100 diseases, either as source or outcome (Pisoschi and Pop, 2015). Therefore, the fact that ROS over-production can kill neurons is well accepted (Brown and Bal-Price, 2003; Taetzsch and Block, 2013). This ROS over-production can occur in the neurons themselves or can also have a glial origin (Yuste et al., 2015).

Mercury Oxidative stress has been implicated in the pathogenesis of methylmercury (MeHg) neurotoxicity. Studies of mature neurons suggest that the mitochondrion may be a major source of MeHg-induced reactive oxygen species and a critical mediator of MeHg-induced neuronal death, likely by activation of apoptotic pathways. (Polunas et al., 2011) (Lu et al., 2011) - MeHg in the mouse cerebrum (in vivo) and in cultured Neuro-2a cells (in vitro). <ul> <li>In vivo - 50µg/kg/day MeHg for 7 consecutive weeks - increased levels of lipid peroxidation in the plasma and cerebral cortex. Decreased GSH level and increase the expressions of caspase-3, -7, and -9, accompanied by Bcl-2 down-regulation and up-regulation of Bax, Bak, and p53.</li> <li>In vitro – 3 and 5 µM MeHg - reduced cell viability, increased oxidative stress damage, and induced several features of mitochondria-dependent apoptotic signals, including increased sub-G1 hypodiploids, mitochondrial dysfunctions, and the activation of PARP, and caspase cascades.  </li> <li>These MeHg-induced apoptotic-related signals could be remarkably reversed by antioxidant NAC.</li> </ul> (Sarafian et al., 1994) - Hypothalamic  mouse neural cell line GT1-7 without and with expression construct for the anti-apoptotic proto-oncogene, bcl-2. <ul> <li>3h exposure, 10 µM MeHg - increased formation of reactive ROS, and decreased levels of GSH, associated with 20% cell death. Cells transfected with an expression construct bcl-2, displayed attenuated ROS induction and negligible cell death.</li> <li>24h exposure, 5 µM MeHg - killed 56% of control cells, but only 19% of bcl-2-transfected cells.</li> <li>By using diethyl maleate to deplete cells of GSH, we demonstrate that the differential sensitivity to MeHg was not due solely to intrinsically different GSH levels. The data suggest that MeHg-mediated cell killing correlates more closely with ROS generation than with GSH levels and that bcl-2 protects MeHg-treated cells by suppressing ROS generation.</li> </ul> (Castoldi et al., 2000) - In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death. <ul> <li>1 hr exposure, 5–10 µM MeHg - impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death.</li> <li>1hr exposure, 0.5–1 µM MeHg - did not compromise cell viability, mitochondrial membrane potential and function at early time points.</li> <li>1hr exposure, 1 µM MeHg - only a small population of neurons (+-20%) dies by necrosis. The surviving neurons show network damage, but maintain membrane integrity, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively display the morphological signs of apoptosis.</li> <li>18hr exposure, 0.5–1 µM MeHg – cells progressively underwent apoptosis reaching the 100% cell death</li> <li>insulin-like growth factor-I partially rescued CGCs from MeHg-triggered apoptosis.</li> </ul> (Kaur,et al., 2006) - primary cell cultures of cerebellar neurons and astrocytes from 7-day-old NMRI mice. 5 mM MeHg for 30 min. <ul> <li>Twenty-one days post-astrocyte isolation - 250mM N-acetyl cysteine (NAC) or 3mM di-ethyl maleate (DEM) added to the wells 12 h prior to MeHg exposure</li> <li>7 days post-neurons isolation - 200mM of NAC or 1.8mM of DEM added to the wells 12 h prior to MeHg exposure</li> <li>The intracellular GSH content was modified by pretreatment with NAC or DEM for 12 h.</li> <li>Treatment with 5 mM Me Hg for 30 min led to significant (p < 0.05) increase in ROS and reduction (p < 0.001) in GSH content.</li> <li>Depletion of intracellular GSH by DEM further increased the generation of MeHg-induced ROS in both cell cultures.</li> <li>NAC supplementation increased intracellular GSH and provided protection against MeHg-induced oxidative stress in both cell cultures.</li> </ul> (Franco et al., 2007) – Mitochondrial enriched fractions from adult (2 months old) Swiss Albino male mice. <ul> <li>MeHg and HgCl2 (10–100 µM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation.</li> <li>Both mercurials induced a significant reduction of GSH in a dose-dependent manner.</li> <li>Correlation analyses showed significant positive correlations between mitochondrial viability and glutathione content for MeHg (Pearson coefficient) 0.933; P < 0.01) and or HgCl2 (Pearson coefficient ) 0.854; P < 0.01).</li> <li>Quercetin (100–300 µM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, which did not display any chelating effect on MeHg or HgCl2, prevented mercurial-induced glutathione oxidation.</li> </ul> (Polunas et al., 2011) - Murine embryonal carcinoma (EC) cells, which differentiate into neurons following exposure to retinoic acid. <ul> <li>4h exposure, 1.5 mM MeHg - earlier and significantly higher levels of ROS production and more extensive mitochondrial depolarization in neurons than in undifferentiated EC cells. cyclosporin A (CsA) completely inhibited mitochondrial depolarization by MeHg in EC cells but only delayed this response in the neurons. In contrast, CsA significantly inhibited MeHg-induced neuronal ROS production. Cyt c release was also more extensive in neurons, with less protection afforded by CsA.</li> </ul> (Sandström et al., 2016) - in vitro 3D human neural tissues from neural progenitor cells derived from human embryonic stem cells. Single MeHg exposure at day 42 of 3D culturing (week 6) and material was collected 72 h after. <ul> <li>1-10 μM - LDH activity increased, confirming induced cell death.</li> <li>5 and 10 μM - increased HMOX1 gene expression as indirect marker of oxidative stress.</li> </ul>   Acrylamide (Allam et al., 2011) - sixty albino Rattus norvegicus, 45 virgin females and 15 mature males. This study examined its effects on the development of external features in cubs. <ul> <li>prenatal intoxicated group - newborns from mothers treated with ACR (10 mg/kg/day by gastric intubation) from day 7 (GD 7) of gestation till birth</li> <li>perinatal intoxicated group - newborns from mothers treated with ACR (10 mg/kg/day by gastric intubation) from GD7 of gestation till D28 after birth</li> </ul> ACR administered either prenatally or perinatally has been shown to induce significant retardation in the new- borns’ body weights development, increase of thiobarbituric acid- reactive substances (TBARS) and oxidative stress (significant reductions in reduced glutathione, total thiols, superoxide dismutase and peroxidase activities) in the developing cerebellum. ACR treatment delayed the proliferation in the granular layer and delayed both cell migration and differentiation. Purkinje cell loss was also seen in acrylamide-treated animals. Ultrastructural studies of Purkinje cells in the perinatal group showed microvacuolations and cell loss. (Lakshmi et al., 2012) - Wistar male albino rats, four groups (n = 6 per group) <ul> <li>II – (Acrylamide) ACR - 30 mg/kg ACR for 30 days: increase in the lipid peroxidative (LPO), protein carbonyl, hydroxyl radical and hydroperoxide levels with subsequent decrease in the activities of enzymic antioxidants and level of GSH. Cortex showed condensed nuclei along with damaged cells. Decrease in the expression of Bcl2 along with simultaneous increase in the expressions of Bax and Bad as compared to control.</li> <li>II rats – ACR + Fish oil -0.5 ml/kg b.w.fish oil orally 10 min before ACR induction with 30 mg/kg for 30 days – reversed significantly all the OS markers.</li> </ul>

Mercury-induced upregulation of GSH level and GR activity as an adaptive mechanism following lactational exposure to methylmercury (10 mg/L in drinking water) associated with motor deficit, suggesting neuronal impairment (Franco et al., 2006).

<table align="center" border="1" cellpadding="0" cellspacing="0" style="width:657px"> <tbody> <tr> <td style="width:103px"> Reference </td> <td style="width:109px"> Chemical Concentration </td> <td style="width:291px"> OS </td> <td style="width:153px"> Cell injury/death </td> </tr> <tr> <td rowspan="3" style="width:103px"> (Sarafian et al., 1994) </td> <td style="width:109px"> MeHg 0 µM </td> <td style="width:291px"> ROS – ±100% DCF Fluorescence GSH – ±150% MCB Fluorescence </td> <td style="width:153px"> ±90% Viability </td> </tr> <tr> <td style="width:109px"> MeHg 5 µM </td> <td style="width:291px"> ROS – ±150% DCF Fluorescence GSH – ±100% MCB Fluorescence </td> <td style="width:153px"> ±80% Viability </td> </tr> <tr> <td style="width:109px"> MeHg 10 µM </td> <td style="width:291px"> ROS – ±200% DCF Fluorescence GSH – ±70% MCB Fluorescence </td> <td style="width:153px"> ±70% Viability </td> </tr> <tr> <td rowspan="5" style="width:103px"> (Lu et al., 2011) in vitro </td> <td style="width:109px"> MeHg 0µM </td> <td style="width:291px"> (2h) ROS – ±100% DCF Fluorescence (24h) 100% intracellular GSH levels </td> <td style="width:153px"> 100% Cell viability </td> </tr> <tr> <td style="width:109px"> MeHg 3µM </td> <td style="width:291px"> (2h) ROS – ±160 DCF Fluorescence (24h) ±60% intracellular GSH levels </td> <td style="width:153px"> ±50% Cell viability </td> </tr> <tr> <td style="width:109px"> MeHg 5µM </td> <td style="width:291px"> (2h) ROS – ±230 DCF Fluorescence  (24h) ±30% intracellular GSH levels </td> <td style="width:153px"> ±10% Cell viability </td> </tr> <tr> <td style="width:109px"> MeHg 3µM + NAC 1mM </td> <td style="width:291px"> (2h) ROS – ±70 DCF Fluorescence (24h) ±90% intracellular GSH levels </td> <td style="width:153px"> ±90% Cell viability </td> </tr> <tr> <td style="width:109px"> MeHg 5µM + NAC 1mM </td> <td style="width:291px"> (2h) ROS% – ±70 DCF Fluorescence (24h) ±90% intracellular GSH levels </td> <td style="width:153px"> ±90% Cell viability </td> </tr> <tr> <td rowspan="6" style="width:103px"> (Kaur et al., 2006) </td> <td style="width:109px"> 0 mM MeHg </td> <td style="width:291px"> (Neurons) GSH – 100v MCB Fluorescence ROS – 100% CMH2DCFDA Fluorescence (Astrocytes) GSH – 100v MCB Fluorescence ROS – 100% CMH2DCFDA Fluorescence </td> <td style="width:153px"> (Neurons) 100% Cell viability (Astrocytes) 100% Cell viability </td> </tr> <tr> <td style="width:109px"> 5 mM MeHg </td> <td style="width:291px"> (Neurons) GSH – ± 50v MCB Fluorescence ROS – ± 400% CMH2DCFDA Fluorescence (Astrocytes) GSH – ± 70% MCB Fluorescence ROS – ± 120% CMH2DCFDA Fluorescence </td> <td style="width:153px"> (Neurons) ±60% Cell viability (Astrocytes) ±75% Cell viability </td> </tr> <tr> <td style="width:109px"> 5 mM MeHg + NAC </td> <td style="width:291px"> (Neurons) GSH – ± 80% MCB Fluorescence ROS – ± 200% CMH2DCFDA Fluorescence (Astrocytes) GSH – ± 80% MCB Fluorescenc e ROS – ± 90% CMH2DCFDA Fluorescence </td> <td style="width:153px"> (Neurons) ±90% Cell viability (Astrocytes) ±90% Cell viability </td> </tr> <tr> <td style="width:109px"> 5 mM MeHg + DEM </td> <td style="width:291px"> (Neurons) GSH – ± 50% MCB Fluorescenc e ROS – ± 470% CMH2DCFDA Fluorescence (Astrocytes) GSH – ± 70% MCB Fluorescence ROS – ± 120% CMH2DCFDA Fluorescence </td> <td style="width:153px"> (Neurons) ±55% Cell viability (Astrocytes) ±65% Cell viability </td> </tr> <tr> <td style="width:109px"> NAC </td> <td style="width:291px"> (Neurons) GSH – ± 110v MCB Fluorescence ROS – ± 100% CMH2DCFDA Fluorescence (Astrocytes) GSH – ±100% MCB Fluorescence ROS – ± 60% CMH2DCFDA Fluorescence </td> <td style="width:153px"> (Neurons) ±110% Cell viability (Astrocytes) ±110% Cell viability </td> </tr> <tr> <td style="width:109px"> DEM </td> <td style="width:291px"> (Neurons) GSH – ± 60% MCB Fluorescence ROS – ± 250% CMH2DCFDA Fluorescence (Astrocytes) GSH – ± 80 MCB Fluorescence ROS – ± 110 CMH2DCFDA Fluorescence </td> <td style="width:153px"> (Neurons) ±80% Cell viability (Astrocytes) ±85% Cell viability </td> </tr> <tr> <td rowspan="4" style="width:103px"> (Franco et al., 2007) </td> <td style="width:109px"> 0 µM MeHg </td> <td style="width:291px"> 100%  GSH </td> <td style="width:153px"> 100% mitochondrial viability </td> </tr> <tr> <td style="width:109px"> 30 µM MeHg </td> <td style="width:291px"> ± 70%  GSH </td> <td style="width:153px"> ± 70%  mitochondrial viability </td> </tr> <tr> <td style="width:109px"> 0 µM HgCl2 </td> <td style="width:291px"> 100% GSH </td> <td style="width:153px"> 100% mitochondrial viability </td> </tr> <tr> <td style="width:109px"> 30 µM HgCl2 </td> <td style="width:291px"> ± 65%  GSH </td> <td style="width:153px"> ± 65% mitochondrial viability </td> </tr> <tr> <td rowspan="4" style="width:103px"> (Lakshmi et al., 2012) </td> <td style="width:109px"> Control </td> <td style="width:291px"> GSH – 0.5 µmoles/mg of protein </td> <td style="width:153px"> ± 6 Damaged cells/Field </td> </tr> <tr> <td style="width:109px"> Acrylamid </td> <td style="width:291px"> GSH – 0.2 µmoles/mg of protein </td> <td style="width:153px"> ± 20 Damaged cells/Field </td> </tr> <tr> <td style="width:109px"> Acrylamid + Fish Oil </td> <td style="width:291px"> GSH – 0.4 µmoles/mg of protein </td> <td style="width:153px"> ± 11 Damaged cells/Field </td> </tr> <tr> <td style="width:109px"> Fish Oil </td> <td style="width:291px"> GSH – 0.5 µmoles/mg of protein </td> <td style="width:153px"> ± 5 Damaged cells/Field </td> </tr> </tbody> </table> <div> </div>

Not Specified Male Not Specified Female

High

All life stages

High High High High

Rat, Mouse: (Sarafian et al., 1994; Castoldi et al., 2000; Kaur et al., 2006; Franco et al., 2007; Lu et al., 2011; Polunas et al., 2011) (Richetti et al., 2011) - Adult and healthy zebrafish of both sexes (12 animals and housed in 3 L) mercury chloride final concentration of 20 mg/L. Mercury chloride promoted a significant decrease in acetylcholinesterase activity and the antioxidant competence was also decreased. (Berntssen, Aatland and Handy, 2003) - Atlantic salmon (Salmo salar L.) were supplemented with mercuric chloride (0, 10, or 100 mg Hg per kg) or methylmercury chloride (0, 5, or 10 mg Hg per kg) for 4 months. Methylmercury chloride <ul> <li>accumulated significantly in the brain of fish fed 5 or 10 mg/kg</li> <li>No mortality or growth reduction</li> <li>- 2-fold increase in the antioxidant enzyme super oxide dismutase (SOD) in the brain</li> <li>10 mg/kg - 7-fold increase of lipid peroxidative products (thiobarbituric acid reactive substances, TBARS) and a subsequently 1.5-fold decrease in anti oxidant enzyme activity (SOD and glutathione peroxidase, GSH-Px). Fish also had pathological damage (vacoulation and necrosis), significantly reduced neural enzyme activity (5-fold reduced monoamine oxidase, MAO, activity), and reduced overall post-feeding activity behaviour.</li> </ul> Mercuric chloride <ul> <li>accumulated significantly in the brain only at 100 mg/kg</li> <li>No mortality or growth reduction</li> <li>100 mg/kg -  significant reduced neural MAO activity and pathological changes (astrocyte proliferation) in the brain, however, neural SOD and GSH-Px enzyme activity, lipid peroxidative products (TBARS), and post feeding behaviour did not differ from controls.</li> </ul>

Allam,  a et al. (2011) ‘Prenatal and perinatal acrylamide disrupts the development of cerebellum in rat: Biochemical and morphological studies.’, Toxicology and industrial health, 27, pp. 291–306. doi: 10.1177/0748233710386412. Bar-Or, D. et al. (2015) ‘Oxidative stress in severe acute illness’, Redox Biology. Elsevier, 4, pp. 340–345. doi: 10.1016/j.redox.2015.01.006. Berntssen, M. H. G., Aatland, A. and Handy, R. D. (2003) ‘Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr’, Aquatic Toxicology, 65(1), pp. 55–72. doi: 10.1016/S0166-445X(03)00104-8. Bhandari, R. and Kuhad, A. (2015) ‘Neuropsychopharmacotherapeutic efficacy of curcumin in experimental paradigm of autism spectrum disorders’, Life Sciences. Elsevier Inc., 141, pp. 156–169. doi: 10.1016/j.lfs.2015.09.012. Brown, G.C. and Bal-Price, A. (2003) ‘Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria’, Molecular Biology, 27(3), pp. 325-355. Castoldi, A. F. et al. (2000) ‘Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury’, Journal of Neuroscience Research, 59(6), pp. 775–787. doi: 10.1002/(SICI)1097-4547(20000315)59:6<775::AID-JNR10>3.0.CO;2-T. Coyle, J. and Puttfarcken, P. (1993) ‘Glutamate Toxicity’, Science, 262, pp. 689–95. Franco, J. L. et al. (2006) ‘Cerebellar thiol status and motor deficit after lactational exposure to methylmercury’, Environmental Research, 102(1), pp. 22–28. doi: 10.1016/j.envres.2006.02.003. Franco, J. L. et al. (2007) ‘Mercurial-induced hydrogen peroxide generation in mouse brain mitochondria: Protective effects of quercetin’, Chemical Research in Toxicology, 20(12), pp. 1919–1926. doi: 10.1021/tx7002323. Gilgun-Sherki, Y., Melamed, E. and Offen, D. (2001) ‘Oxidative stress induced-neurodegenerative diseases: The need for antioxidants that penetrate the blood brain barrier’, Neuropharmacology, 40(8), pp. 959–975. doi: 10.1016/S0028-3908(01)00019-3. Grandjean, P. and Landrigan, P. J. (2014) ‘Neurobehavioural effects of developmental toxicity’, The Lancet Neurology, 13(3), pp. 330–338. doi: 10.1016/S1474-4422(13)70278-3. Kaur, P., Aschner, M. and Syversen, T. (2006) ‘Glutathione modulation influences methyl mercury induced neurotoxicity in primary cell cultures of neurons and astrocytes’, NeuroToxicology, 27(4), pp. 492–500. doi: 10.1016/j.neuro.2006.01.010. Lakshmi, D. et al. (2012) ‘Ameliorating effect of fish oil on acrylamide induced oxidative stress and neuronal apoptosis in cerebral cortex’, Neurochemical Research, 37(9), pp. 1859–1867. doi: 10.1007/s11064-012-0794-1. Lu, T. H. et al. (2011) ‘Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury’, Toxicology Letters. Elsevier Ireland Ltd, 204(1), pp. 71–80. doi: 10.1016/j.toxlet.2011.04.013. Madhavan, L. et al. (2006) ‘Increased "vigilance" of antioxidant mechanisms in neural stem cells potentiates their capability to resist oxidative stress’, Stem Cells 24(2) pp. 2110-2119. Markesbery, W. R. (1997) ‘Oxidative stress hypothesis in Alzheimer’s disease’, Free Radical Biology and Medicine, 23(1), pp. 134–147. doi: 10.1016/S0891-5849(96)00629-6. Pisoschi, A. M. and Pop, A. (2015) ‘The role of antioxidants in the chemistry of oxidative stress: A review’, European Journal of Medicinal Chemistry. Elsevier Masson SAS, 97, pp. 55–74. doi: 10.1016/j.ejmech.2015.04.040. Polunas, M. et al. (2011) ‘Role of oxidative stress and the mitochondrial permeability transition in methylmercury cytotoxicity’, NeuroToxicology. Elsevier B.V., 32(5), pp. 526–534. doi: 10.1016/j.neuro.2011.07.006. Reynolds, A. et al. (2007) ‘Oxidative Stress and the Pathogenesis of Neurodegenerative Disorders’, International Review of Neurobiology, 82(7), pp. 297–325. doi: 10.1016/S0074-7742(07)82016-2. Richetti, S. K. et al. (2011) ‘Acetylcholinesterase activity and antioxidant capacity of zebrafish brain is altered by heavy metal exposure’, NeuroToxicology. Elsevier B.V., 32(1), pp. 116–122. doi: 10.1016/j.neuro.2010.11.001. Rossignol, D. A. and Frye, R. E. (2014) ‘Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism’, Frontiers in Physiology, 5 APR(April), pp. 1–15. doi: 10.3389/fphys.2014.00150. Sandström, J. et al. (2016) ‘Toxicology in Vitro Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing’, Tiv, pp. 1–12. doi: 10.1016/j.tiv.2016.10.001. Sandström, J. et al. (2017) ‘Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures’, NeuroToxicology, 60, pp. 116–124. doi: 10.1016/j.neuro.2017.04.010. Sarafian, T. A. et al. (1994) ‘Bcl-2 Expression Decreases Methyle Mercury-Induced Free-Radical Generation and Cell Killing in a Neural Cell Line’, Toxicol. Lett., 74(2), pp. 149–155. Sies, H. (2015) ‘Oxidative stress: A concept in redox biology and medicine’, Redox Biology. Elsevier, 4, pp. 180–183. doi: 10.1016/j.redox.2015.01.002. Taetzsch, T. and Block, M.L. (2013) ‘Pesticides, microglial NOX2, and Parkinson's disease’, J Biochem Molecular Toxicology, 27(2), pp. 137-149. Wang, X. and Michaelis, E. K. (2010) ‘Selective neuronal vulnerability to oxidative stress in the brain’, Frontiers in Aging Neuroscience, 2(MAR), pp. 1–13. doi: 10.3389/fnagi.2010.00012. Wells, P. G. et al. (2009) ‘Oxidative stress in developmental origins of disease: Teratogenesis, neurodevelopmental deficits, and cancer’, Toxicological Sciences, 108(1), pp. 4–18. doi: 10.1093/toxsci/kfn263. Yuste, J.E., et al., 2015. Implications of glial nitric oxide in neurodegenerative diseases. Front Cell Neurosci. 9, 322. AOPWiki 2017-11-09T04:13:46

2020-02-07T09:32:24

<upstream-id>ee9118a3-6ea8-4f74-982c-75680fa3ba62</upstream-id> <downstream-id>467f3406-99f1-4b9a-a479-96b50d507863</downstream-id> Damaged hepatocytes release reactive oxygen species (ROS), cytokines such as TGF-β1 and TNF-α, and chemokines which lead to oxidative stress, inflammatory signalling and finally activation of Kupffer cells (KCs). ROS generation in hepatocytes results from oxidative metabolism by NADH oxidase (NOX) and cytochrome 2E1 activation as well as through lipid peroxidation. Damaged liver cells trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (DAMPs), which activate KCs through toll-like receptors and recruit activated neutrophils and monocytes into the liver. Central to this inflammatory response is the promotion of ROS formation by these phagocytes. Upon initiation of apoptosis hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies; these apoptotic bodies are consecutively engulfed by KCs and cause their activation. This increased phagocytic activity strongly up-regulates NOX expression in KCs, a superoxide producing enzyme of phagocytes with profibrogenic activity, as well as nitric oxide synthase (iNOS) mRNA transcriptional levels with consequent harmful reaction between ROS and nitricoxide (NO), like the generation of cytotoxic peroxinitrite (N2O3). ROS and/or diffusible aldehydes also derive from liver sinusoidal endothelial cells (LSECs) which are additional initial triggers of KC activation. <a href="#cite_note-Winwood_1993-1">[1]</a> <a href="#cite_note-Luckey_2001-2">[2]</a> <a href="#cite_note-Roberts_2007-3">[3]</a> <a href="#cite_note-Malhi_2010-4">[4]</a> <a href="#cite_note-Canbay_2004-5">[5]</a> <a href="#cite_note-Orrenius_2011-6">[6]</a> <a href="#cite_note-Kolios_2006-7">[7]</a> <a href="#cite_note-Kisseleva-8">[8]</a> <a href="#cite_note-Jaeschke_2011-9">[9]</a> <a href="#cite_note-Li_2008-10">[10]</a> <a href="#cite_note-Poli_2000-11">[11]</a>

There is a functional relationship between cell injury/death and KC activation, consistent with established biological knowledge. <a href="#cite_note-Winwood_1993-1">[1]</a> <a href="#cite_note-Luckey_2001-2">[2]</a> <a href="#cite_note-Roberts_2007-3">[3]</a> <a href="#cite_note-Malhi_2010-4">[4]</a> <a href="#cite_note-Canbay_2004-5">[5]</a> <a href="#cite_note-Orrenius_2011-6">[6]</a> <a href="#cite_note-Kolios_2006-7">[7]</a> <a href="#cite_note-Kisseleva-8">[8]</a> <a href="#cite_note-Jaeschke_2011-9">[9]</a> <a href="#cite_note-Li_2008-10">[10]</a> <a href="#cite_note-Poli_2000-11">[11]</a>

There is convincing theoretical evidence that hepatocyte injury and apoptosis causes KC activation, as well as inflammation and oxidative stress. But there are only limited experimental studies which could show that there is a direct relationship between these two events with temporal concordance. Specific markers for activated KCs have not been identified yet. KC activation cannot be detected morphologically by staining techniques since cell morphology does not change, but cytokines release can be measured (with the caveat that KCs activate spontaneously in vitro) and used as marker for KC activation.<a href="#cite_note-12">[12]</a><a href="#cite_note-13">[13]</a> Tukov et al. examined the effects of KCs cultured in contact with rat hepatocytes. They found that by adding KCs to the cultures they could mimic in vivo drug-induced inflammatory responses. Experiments on cells of the macrophage lineage showed significant aldehyde-induced stimulation of the activity of protein kinase C, an enzyme involved in several signal transduction pathways. Further, 4-Hydroxynonenal (HNE) was demonstrated to up-regulate TGF-β1 expression and synthesis in isolated rat KCs.<a href="#cite_note-Tukov_2006-14">[14]</a> Canbay et al could prove that engulfment of hepatocyte apoptotic bodies stimulated KC generation of cytokines. <a href="#cite_note-15">[15]</a>

The detailed mechanisms of the KC - hepatocyte interaction and its consequences for both normal and toxicant-driven liver responses remain to be determined. KC activation followed by cytokine release is associated in some cases with evident liver damage, whereas in others this event is unrelated to liver damage or may be even protective; apparently this impact is dependent on the quantity of KC activation; excessive or prolonged release of KC mediators can switch an initially protective mechanism to a damaging inflammatory response. Evidence suggests that low levels of cytokine release from KCs constitute a survival signal that protects hepatocytes from cell death and in some cases, stimulates proliferation. <a href="#cite_note-Roberts_2007-3">[3]</a>

no quantitative data

High High

Human: <a href="#cite_note-Winwood_1993-1">[1]</a><a href="#cite_note-Roberts_2007-3">[3]</a><a href="#cite_note-Kolios_2006-7">[7]</a> Rat: <a href="#cite_note-Tukov_2006-14">[14]</a><a href="#cite_note-Roberts_2007-3">[3]</a>

<ol class="references"> <li id="cite_note-Winwood_1993-1">↑ <a href="#cite_ref-Winwood_1993_1-0">1.0</a> <a href="#cite_ref-Winwood_1993_1-1">1.1</a> <a href="#cite_ref-Winwood_1993_1-2">1.2</a> Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59. </li> <li id="cite_note-Luckey_2001-2">↑ <a href="#cite_ref-Luckey_2001_2-0">2.0</a> <a href="#cite_ref-Luckey_2001_2-1">2.1</a> Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240. </li> <li id="cite_note-Roberts_2007-3">↑ <a href="#cite_ref-Roberts_2007_3-0">3.0</a> <a href="#cite_ref-Roberts_2007_3-1">3.1</a> <a href="#cite_ref-Roberts_2007_3-2">3.2</a> <a href="#cite_ref-Roberts_2007_3-3">3.3</a> <a href="#cite_ref-Roberts_2007_3-4">3.4</a> Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15. </li> <li id="cite_note-Malhi_2010-4">↑ <a href="#cite_ref-Malhi_2010_4-0">4.0</a> <a href="#cite_ref-Malhi_2010_4-1">4.1</a> Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194. </li> <li id="cite_note-Canbay_2004-5">↑ <a href="#cite_ref-Canbay_2004_5-0">5.0</a> <a href="#cite_ref-Canbay_2004_5-1">5.1</a> Canbay, A., S.L. Friedman and G.J. Gores (2004), Apoptosis: the nexus of liver injury and fibrosis, Hepatology, vol. 39, no. 2, pp. 273-278. </li> <li id="cite_note-Orrenius_2011-6">↑ <a href="#cite_ref-Orrenius_2011_6-0">6.0</a> <a href="#cite_ref-Orrenius_2011_6-1">6.1</a> Orrenius, S., P. Nicotera and B. Zhivotovsky (2011), Cell death mechanisms and their implications in toxicology, Toxicol. Sci, vol. 119, no. 1, pp. 3-19. </li> <li id="cite_note-Kolios_2006-7">↑ <a href="#cite_ref-Kolios_2006_7-0">7.0</a> <a href="#cite_ref-Kolios_2006_7-1">7.1</a> <a href="#cite_ref-Kolios_2006_7-2">7.2</a> Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420. </li> <li id="cite_note-Kisseleva-8">↑ <a href="#cite_ref-Kisseleva_8-0">8.0</a> <a href="#cite_ref-Kisseleva_8-1">8.1</a> Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122. </li> <li id="cite_note-Jaeschke_2011-9">↑ <a href="#cite_ref-Jaeschke_2011_9-0">9.0</a> <a href="#cite_ref-Jaeschke_2011_9-1">9.1</a> Jaeschke, H. (2011), Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts, J Gastroenterol Hepatol. vol. 26, suppl. 1, pp. 173-179. </li> <li id="cite_note-Li_2008-10">↑ <a href="#cite_ref-Li_2008_10-0">10.0</a> <a href="#cite_ref-Li_2008_10-1">10.1</a> Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428. </li> <li id="cite_note-Poli_2000-11">↑ <a href="#cite_ref-Poli_2000_11-0">11.0</a> <a href="#cite_ref-Poli_2000_11-1">11.1</a> Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98. </li> <li id="cite_note-12"><a href="#cite_ref-12">↑</a> Canbay, A. et al. (2003), Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression, Hepatology, vol. 38, no. 5, pp. 1188-1198. </li> <li id="cite_note-13"><a href="#cite_ref-13">↑</a> Soldatow, V.Y. et al. (2013), In vitro models for liver toxicity testing, Toxicol Res, vol. 2, no.1, pp. 23–39. </li> <li id="cite_note-Tukov_2006-14">↑ <a href="#cite_ref-Tukov_2006_14-0">14.0</a> <a href="#cite_ref-Tukov_2006_14-1">14.1</a> Tukov, F.F. et al. (2006), Modeling inflammation-drug interactions in vitro: a rat Kupffer cell-hepatocyte co-culture system, Toxicol In Vitro, vol. 20, no. 8, pp. 1488-1499. </li> <li id="cite_note-15"><a href="#cite_ref-15">↑</a> LeCluyse, E.L. et al. (2012), Organotypic liver culture models: meeting current challenges in toxicity testing, Crit Rev Toxicol, vol. 42, no. 6, 501-548. </li> </ol> AOPWiki 2016-11-29T18:41:33

2016-11-29T19:54:53

<upstream-id>467f3406-99f1-4b9a-a479-96b50d507863</upstream-id> <downstream-id>4cbabf15-ee16-4b8c-bcba-d8f23b4f39bd</downstream-id>

AOPWiki 2026-02-10T09:00:33

2026-02-10T09:00:33

<upstream-id>4cbabf15-ee16-4b8c-bcba-d8f23b4f39bd</upstream-id> <downstream-id>c52c0905-148d-48dc-b798-f79070cd2a84</downstream-id>

AOPWiki 2026-02-10T09:00:43

2026-02-10T09:00:43

<upstream-id>c52c0905-148d-48dc-b798-f79070cd2a84</upstream-id> <downstream-id>358d0a72-b27a-44eb-b6ac-1e9303d0c472</downstream-id>

AOPWiki 2026-02-10T09:01:01

2026-02-10T09:01:01

<upstream-id>358d0a72-b27a-44eb-b6ac-1e9303d0c472</upstream-id> <downstream-id>584ff836-d2e7-4952-a1b9-53934ce32a59</downstream-id> Up-regulation of collagen synthesis following hepatic stellate cell (HSC) activation is among the most striking molecular responses of HSCs to injury and is mediated by both transcriptional and post-transcriptional mechanisms. Activated HSCs do not only proliferate and increase cell number, but also increase collagen production per cell. Synthesis of type I collagen is initiated by expression of the col1a1 and col1a2 genes, giving rise to α 1(I) and α 2(I) procollagen mRNAs in a 2:1 ratio. Upon activation of HSCs and other myofibroblast precursors, there is a > 50-fold increase in α 1(I) procollagen mRNA levels. The half-life of collagen α1(I) mRNA increases 20-fold in activated HSCs compared with quiescent HSCs. Monocytes and macrophages are involved in inflammatory actions by producing large amounts of Nitric oxide (NO) and inflammatory cytokines such as TNF-α which have a direct stimulatory effect on HSC collagen synthesis. Synthesis of TGF-α and TGF-β promotes activation of neighbouring quiescent HSCs, whereas the release of HGF (hepatocyte growth factor) stimulates regeneration of adjacent hepatocytes. The basement membrane-like matrix is normally comprised of collagens IV and VI, which is progressively replaced by collagens I and III and cellular fibronectin during fibrogenesis. Although multiple extracellular matrix (ECM) components are up-regulated, type I collagen is the most abundant protein. These changes in ECM composition initiate several positive feedback pathways that further amplify collagen production. Increasing matrix stiffness is a stimulus for HSC activation and matrix-provoked signals link to other growth factor receptors through integrin-linked kinase and transduce via membrane-bound guanosine triphosphate binding proteins, in particular Rho67 and Rac, signals to the actin cytoskeleton that promote migration and contraction. The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Down-regulated expression of degrading Matrix metalloproteinases (MMPs) and up-regulation of tissue inhibitors of metalloproteinases (TIMPs), MMP- inhibitors, lead to a net decrease in protease activity, and therefore, matrix accumulation. Chronic inflammation, hypoxia and oxidative stress reactivate epithelial-mesenchymal transition (EMT) developmental programmes that converge in the activation of NF-kB. Cells that may transdifferentiate into fibrogenic myofibroblasts are hepatocytes and cholangiocytes. Additional sources of ECM include bone marrow (which probably gives rise to circulating fibrocytes) and portal fibroblasts (Benyon and Arthur; 2001; Milani et al., 1994; Safadi and Friedman, 2002; Kolios et al.,2006; Bataller and Brenner, 2005; Lee und Friedman 2011; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008;  Kershenobich Stalnikowitz and Weisssbrod , 2003; López-Novoa and Nieto, 2009; Friedman, 2010; 2008; Dalton et al., 2009; Leung, et al., 2008; Nan et al., 2013;  Hamdy and El-Demerdash, 2012;Li, Li et al., 2012; Natajaran et al., 2006; Luckey and Petersen, 2001;  Chen and Raghunath, 2009;Thompson et al., 2011; Henderson and Iredale, 2007).

There is general acceptance that HSCs are collagen producing cells and key actors in fibrogenesis. The functional relationship between these KEs is consistent with biological knowledge (Benyon and Arthur; 2001; Milani et al., 1994; Safadi and Friedman, 2002; Kolios et al.,2006; Bataller and Brenner, 2005; Lee und Friedman 2011; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008;  Kershenobich Stalnikowitz and Weisssbrod , 2003; López-Novoa and Nieto, 2009).

It is difficult to stimulate sufficient collagen production and its subsequent incorporation into a pericellular matrix in vitro; therefore analytical methods have focused on measurement of pro-collagen secreted into culture medium or measurement of α-smooth muscle actin (α-SMA) expression, a marker of fibroblast activation. In primary culture, HSCs from normal liver begin to express α-SMA coincident with culture-induced activation ( Chen and Raghunath, 2009; Rockey et al.,1992).

no inconsistencies

no quantitative data

Not Specified Unspecific

Not Specified

All life stages

High High

Human: Safadi and Friedman, 2002; Bataller and Brenner, 2005; Lee und Friedman 2011. Rat: Li, Li et al., 2012; Luckey and Petersen, 2001;  Rockey et al., 1992

<ul> <li>Benyon, R.C. and M.J. Arthur (2001), Extracellular matrix degradation and the role of stellate cells, Semin Liver Dis, vol. 21, no. 3, pp. 373-384.</li> <li>Milani, S. et al. (1994), Differential expression of matrix-metalloproteinase-1 and -2 genes in normal and fibrotic human liver, Am J Pathol, vol. 144, no. 3, pp. 528-537.</li> <li>↑Safadi, R. and S.L. Friedman (2002), Hepatic fibrosis--role of hepatic stellate cell activation, MedGenMed, vol 4, no. 3, p. 27.</li> <li>Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.</li> <li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li> <li>Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract Res Clin Gastroenterol, vol. 25, no. 2, pp. 195-206.</li> <li>Guo, J. and S. L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</li> <li>Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.</li> <li>Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.</li> <li>Kershenobich Stalnikowitz, D. and A.B. Weisssbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</li> <li>López-Novoa, J.M. and M.A. Nieto (2009), Inflammation and EMT: an alliance towards organ fibrosis and cancer progression, EMBO Mol Med, vol. 1. no. 6-7, pp. 303–314.</li> <li>Friedman, S.L (2010), Evolving challenges in hepatic fibrosis, Nat. Rev. Gastroenterol. Hepatol, vol. 7, no. 8, pp. 425–436.</li> <li>Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.</li> <li>Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.</li> <li>Leung, T.M. et al. (2008), Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis, Int J Exp Pathol, vol. 89, no. 4, pp. 241-250.</li> <li>Nan, Y.M. et al. (2013), Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice, Lipids in Health and Disease, vol. 12, p. 11.</li> <li>Hamdy, N. and E. El-Demerdash. (2012), New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage, Toxicol Appl Pharmacol, vol. 261, no. 3, pp. 292-299.</li> <li>Li, Li et al. (2012), Establishment of a standardized liver fibrosis model with different pathological stages in rats, Gastroenterol Res Pract; vol. 2012, Article ID 560345.</li> <li>Natajaran, S.K. et al. (2006), Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J Gastroenterol Hepatol, vol. 21, no. 6, pp. 947-957.</li> <li>Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</li> <li>Chen, C. and M. Raghunath (2009), Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art, Fibrogenesis Tissue Repair, vol. 15, no. 2, p. 7.</li> <li>Thompson, K.J., I.H. McKillop and L.W. Schrum (2011), Targeting collagen expression in alcoholic liver disease, World J Gastroenterol, vol. 17, no. 20, pp. 2473-2481.</li> <li>Henderson, N.C. and J.P. Iredale (2007), Liver fibrosis: cellular mechanisms of progression and resolution, Clin Sci (Lond), vol. 112, no. 5, pp. 265-280.</li> <li>Rockey, D.C. et al. (1992), Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture, J Submicrosc Cytol Pathol, vol. 24, no. 2, pp. 193-203.</li> </ul> AOPWiki 2016-11-29T18:41:33

2018-12-05T08:51:54

<upstream-id>584ff836-d2e7-4952-a1b9-53934ce32a59</upstream-id> <downstream-id>b9b585e5-9eb2-479f-88ff-b73ad695cc37</downstream-id> Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) proteins including collagen. Liver fibrosis results from an imbalance between the deposition and degradation of ECM and a change of ECM composition; the latter initiates several positive feedback pathways that further amplify fibrosis. With chronic injury, there is progressive substitution of the liver parenchyma by scar tissue. Deposition of collagen in the liver progressively disrupts the normal hepatic architecture so that the normal relationship between vascular inflow and outflow is destroyed and the normal collagen content around hepatic sinusoids in regenerating nodules becomes modified.Advanced liver fibrosis results in cirrhosis (Lee and Friedman, 2011; Bataller and Brenner, 2005; Pellicoro et al., 2014;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997).

By definition, liver fibrosis is the excessive accumulation of ECM proteins that are produced by HSCs. The KER between this KE and the AO is undisputed (Lee and Friedman, 2011; Bataller and Brenner, 2005;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997).

There is a smooth transition from ECM accumulation to liver fibrosis without a definite threshold and plenty in vivo evidence exists that ECM accumulation is a pre-stage of liver fibrosis (Lee and Friedman, 2011; Bataller and Brenner, 2005;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997).

no inconsistencies

no quantitative data

Not Specified Unspecific

Not Specified

All life stages

High High

Human: Lee and Friedman, 2011; Bataller and Brenner, 2005; Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997.   Rat :Liedtke et al., 2013.

<ul> <li>Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract Res Clin Gastroenterol, vol. 25, no. 2, pp. 195-206.</li> <li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li> <li>Pellicoro, A. et al. (2014), Liver fibrosis and repair: immune regulation of wound healing in a solid organ, Nat Rev Immunol, vol. 14, no. 3, pp. 181-194.</li> <li>Brancatelli, G. et al. (2009), Focal confluent fibrosis in cirrhotic liver: natural history studied with serial CT, AJR Am J Roentgenol, vol. 192, no. 5, pp. 1341-1347.</li> <li>Rockey, D.C. and S.L. Friedman (2006), Hepatic fibrosis and cirrhosis, Zakim and Boyer's Hepatology, 5th edition, section 1, chapter 6, pp. 87-109.</li> <li>Poynard, T., P. Bedossa and P. Opolon (1997), Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups, Lancet, vol. 349, no. 9055, pp. 825-832.</li> <li>Liedtke, C. et al. (2013), Experimental liver fibrosis research: update on animal models, legal issues and translational aspects, Fibrogenesis Tissue Repair, vol. 6, no. 1, p. 19.</li> </ul> AOPWiki 2016-11-29T18:41:33

2018-12-05T08:52:45

<upstream-id>b9b585e5-9eb2-479f-88ff-b73ad695cc37</upstream-id> <downstream-id>f04982e3-3da4-4c68-a176-ccec6802325f</downstream-id>

AOPWiki 2026-02-10T09:02:04

2026-02-10T09:02:04

<upstream-id>f04982e3-3da4-4c68-a176-ccec6802325f</upstream-id> <downstream-id>82127612-bb0f-4696-988e-0bc50963ee3e</downstream-id>

AOPWiki 2026-02-10T09:02:13

2026-02-10T09:02:13

Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress

Increased 11β-HSD1 leading to MASLD via DNL-associated oxidative stress

You Song

You Song1, Jorke H. Kamstra2, Matej Oresic3,4 1 Norwegian Institute for Water Research, Økernveien 94, Oslo, Norway 2 Utrecht University, Institute for Risk Assessment Sciences (IRAS), Utrecht, the Netherlands 3 Örebro University, School of Medical Sciences, Örebro, Sweden 4 University of Turku, Turku Bioscience Centre, Turku, Finland   Acknowledgement: This project was supported by the “Investigation of endocrine-disrupting chemicals as contributors to progression of metabolic dysfunction-associated steatotic liver disease” (EDC-MASLD) consortium funded by the Horizon Europe Program of the European Union (Grant Agreement 101136259).  Shihori Tanabe

BY-SA

Under Development

2.7

This adverse outcome pathway (AOP) describes a mechanistic sequence linking altered glucocorticoid receptor (GR) signaling to the progression of metabolic dysfunction–associated steatotic liver disease (MASLD) through impaired hepatic very-low-density lipoprotein (VLDL) export and subsequent endoplasmic reticulum (ER) stress. Disruption of GR signaling reduces VLDL assembly and secretion, leading to intrahepatic lipid retention and hepatocellular lipotoxicity. Accumulation of lipids within the ER overwhelms protein and lipid handling capacity, inducing ER stress and maladaptive unfolded protein response signaling. Sustained ER stress promotes hepatocyte injury, inflammatory activation, and profibrotic signaling, including TGF-β–mediated hepatic stellate cell activation. These processes drive disease progression from steatosis to steatohepatitis (MASH), fibrosis, and ultimately cirrhosis. This AOP provides a biologically plausible and regulatory-relevant framework for identifying endocrine-disrupting chemicals (EDCs) that promote MASLD progression through GR-mediated disruption of hepatic lipid export and ER homeostasis. Efficient export of triglycerides via VLDL is essential for maintaining hepatocellular lipid balance and preventing intracellular lipid overload. Impairment of VLDL assembly or secretion results in lipid accumulation within hepatocytes, particularly within the endoplasmic reticulum, where lipoprotein assembly occurs. This accumulation can directly disrupt ER membrane integrity and protein folding capacity, leading to ER stress. Glucocorticoid receptor (GR) signaling regulates multiple aspects of hepatic lipid metabolism, including lipoprotein assembly, apolipoprotein expression, and triglyceride trafficking. Altered GR signaling—whether through dysregulation or chemical interference—can suppress VLDL export independently of changes in lipid influx or insulin sensitivity. This AOP was developed to capture ER stress as a direct consequence of impaired lipid export, providing a mechanistically distinct pathway linking GR disruption to MASLD progression.

This AOP was developed using an expert-driven conceptual framework supported by targeted literature evaluation across endocrinology, hepatic lipid metabolism, ER stress biology, and chronic liver disease. Initial scoping identified reduced VLDL export as a key mechanistic driver of ER stress and downstream liver injury. Focused literature searches were conducted to identify evidence supporting: <ul> <li> GR regulation of VLDL assembly and secretion </li> <li> Effects of impaired VLDL export on hepatic lipid retention </li> <li> Induction of ER stress by intracellular lipid accumulation </li> <li> ER stress–mediated hepatocyte injury and inflammatory signaling </li> <li> Fibrogenic pathways involving TGF-β signaling and hepatic stellate cell activation </li> </ul> Evidence from human clinical studies, animal models, and mechanistic in vitro systems was prioritized, with emphasis on chronic perturbations relevant to endocrine disruption.

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

Steatohepatitis represents a clinically recognized and pathologically defined stage of metabolic dysfunction–associated steatotic liver disease (MASLD), characterized by hepatic steatosis accompanied by hepatocellular injury and inflammation. It is a critical transition point between reversible metabolic steatosis and progressive, potentially irreversible liver pathology, including fibrosis, cirrhosis, and hepatocellular carcinoma. As such, an increase in steatohepatitis severity constitutes a biologically meaningful and adverse health outcome. <h2>Human Health Relevance</h2> Steatohepatitis (formerly NASH; now MASH under MASLD nomenclature) is associated with: <ul> <li> Increased risk of liver fibrosis progression </li> <li> Elevated liver-related morbidity and mortality </li> <li> Increased risk of hepatocellular carcinoma </li> <li> Higher overall cardiometabolic mortality </li> </ul> Histologically confirmed steatohepatitis is a strong predictor of disease progression compared to simple steatosis. Therefore, regulatory concern is substantially higher once inflammatory and hepatocellular injury components are present. <h2>Scientific Basis for Domain of Applicability</h2> <h3>Taxonomic Applicability</h3> The adverse outcome is highly relevant to mammals, particularly humans, due to conserved hepatic architecture, lipid metabolism, inflammatory signaling, and fibrogenic pathways. Rodent models (e.g., high-fat diet, Western diet, glucocorticoid exposure models) reliably reproduce key histopathological features of steatohepatitis, including: <ul> <li> Steatosis with hepatocyte ballooning </li> <li> Lobular inflammation </li> <li> Early perisinusoidal fibrosis </li> </ul> This cross-species concordance supports high biological plausibility within Mammalia. <h3>Life Stage Applicability</h3> The adverse outcome is most relevant in adolescent and adult life stages, where metabolic systems are fully developed and chronic exposure conditions can lead to progressive disease. While pediatric MASLD exists, the majority of mechanistic and regulatory evidence derives from adult populations and adult rodent models. <h3>Sex Applicability</h3> Steatohepatitis occurs in both males and females. Sex differences in susceptibility and progression rate have been reported, likely reflecting hormonal influences on lipid metabolism and inflammation. However, the pathological entity and its progression mechanisms are conserved across sexes. <h2>Weight of Evidence for Adversity</h2> The weight of evidence supporting steatohepatitis as an adverse outcome is strong based on: <ol> <li> Clinical Evidence <ul> <li> Histologically confirmed steatohepatitis predicts fibrosis progression and mortality. </li> <li> Longitudinal human studies demonstrate increased liver-related outcomes compared to simple steatosis. </li> </ul> </li> <li> Pathophysiological Evidence <ul> <li> Hepatocyte ballooning reflects cellular injury and cytoskeletal disruption. </li> <li> Inflammatory infiltration drives sustained tissue damage and fibrogenesis. </li> <li> Cytokine and TGF-β signaling link inflammation directly to fibrosis progression. </li> </ul> </li> <li> Consistency Across Models <ul> <li> Reproducible induction in multiple rodent models. </li> <li> Mechanistic concordance between experimental systems and human disease. </li> </ul> </li> <li> Irreversibility Consideration <ul> <li> While early steatohepatitis may be partially reversible, sustained inflammation significantly increases the probability of irreversible fibrosis. </li> </ul> </li> </ol> <h2>Regulatory Relevance</h2> An increase in steatohepatitis severity represents: <ul> <li> A clear adverse effect at the organ level </li> <li> A progression beyond adaptive metabolic perturbation </li> <li> A disease-defining pathological state recognized in clinical practice </li> </ul> From a regulatory perspective, this adverse outcome is relevant for: <ul> <li> Hazard identification </li> <li> Chronic toxicity assessment </li> <li> Endocrine and metabolic disruptor evaluation </li> <li> Integration into adverse outcome pathways supporting chemical prioritization </li> </ul> Because steatohepatitis is a well-defined diagnostic and pathological entity with established clinical consequences, it provides a robust anchor for AOP-based risk assessment frameworks.

From the OECD - GUIDANCE DOCUMENT ON DEVELOPING AND ASSESSING ADVERSE OUTCOME PATHWAYS - Series on Testing and Assessment 18: "...an adverse effect that is of regulatory interest (e.g. repeated dose liver fibrosis)"

An increase in cirrhosis represents: <ul> <li> A severe, adverse organ-level outcome </li> <li> Largely irreversible structural liver damage </li> <li> A major determinant of liver-related morbidity and mortality </li> </ul> From a regulatory perspective, cirrhosis constitutes a high-concern adverse outcome suitable for: <ul> <li> Chronic toxicity hazard identification </li> <li> Chemical prioritization </li> <li> Risk assessment frameworks </li> </ul> Because cirrhosis reflects irreversible architectural and functional liver impairment, it anchors the most severe end of MASLD progression within the AOP network.

adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified Not Specified Not Specified

This AOP is biologically plausible and supported by moderate to strong empirical evidence linking impaired hepatic lipid export to ER stress and progressive liver injury. The sequence of key events reflects conserved cellular stress responses and fibrogenic mechanisms observed across mammalian species. The AOP is particularly well suited for hazard identification and prioritization of chemicals that disrupt GR-regulated lipid handling without necessarily inducing insulin resistance or increased lipid influx. It complements other GR-mediated MASLD AOPs by highlighting ER stress as a downstream consequence of defective VLDL export. <ul> <li> Taxa: Mammals (humans and laboratory rodents) </li> <li> Life stage: Primarily adolescents and adults </li> <li> Sex: Applicable to both sexes; sex-dependent differences may occur due to hormonal modulation of lipid metabolism </li> <li> Biological context: Chronic endocrine perturbation, impaired lipoprotein metabolism, metabolic stress </li> </ul> This AOP is not intended to describe acute hepatotoxicity and is most applicable to chronic exposure scenarios.

Evidence supporting the essentiality of the key events includes: <ul> <li> Altered GR signaling: Experimental modulation of GR activity affects hepatic lipid handling and VLDL secretion. </li> <li> Reduced VLDL export: Genetic or pharmacological impairment of VLDL assembly leads to hepatic lipid accumulation and ER stress. </li> <li> ER stress: Attenuation of ER stress responses reduces hepatocyte injury and inflammatory signaling in MASLD models. </li> <li> Inflammatory and fibrogenic activation: Inhibition of Kupffer cell activation, hepatic stellate cell activation, or TGF-β signaling mitigates fibrosis progression. </li> </ul> Together, these findings support the causal role of each KE in driving downstream MASLD outcomes.

Across the KERs in this AOP: <ul> <li> Biological plausibility is strong, based on established roles of VLDL export in hepatocyte lipid and ER homeostasis. </li> <li> Empirical support is moderate to strong, with consistent evidence across in vivo and in vitro systems. </li> <li> Quantitative understanding is limited, particularly regarding thresholds for ER stress induction following impaired lipid export. </li> </ul> Overall, the weight of evidence supports confidence in the pathway for regulatory-relevant applications.

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td>Dietary lipid load</td> <td>Exacerbates lipid accumulation when VLDL export is impaired</td> <td>VLDL export ↓ → ER stress</td> </tr> <tr> <td>Apolipoprotein availability</td> <td>Modulates efficiency of VLDL assembly</td> <td>GR signaling → VLDL export</td> </tr> <tr> <td>ER chaperone capacity</td> <td>Influences resilience to lipid-induced ER stress</td> <td>ER stress → cell injury</td> </tr> <tr> <td>Inflammatory milieu</td> <td>Amplifies hepatocyte injury and fibrogenesis</td> <td>Cell injury → fibrosis</td> </tr> </tbody> </table>

Quantitative data exist for relationships between impaired VLDL export, hepatic lipid accumulation, and ER stress marker induction. However, quantitative integration across downstream inflammatory and fibrotic events remains limited. Accordingly, this AOP is best applied qualitatively or semi-quantitatively.

This AOP may support: <ul> <li> Identification of GR-modulating chemicals that impair hepatic lipid export </li> <li> Integration of ER stress endpoints into MASLD-relevant testing strategies </li> <li> Complementary assessment of ER-centric mechanisms alongside mitochondrial stress pathways </li> <li> Construction of AOP networks capturing convergent routes to MASLD progression </li> </ul>

AOPWiki 2026-02-10T08:28:31

2026-02-26T04:15:39