Prolonged TLR9 activation

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

1403-66-3

Gentamicin

Gentacycol Gentalline gentamicina gentamicine GENTAMYCIN Gentavet Lyramycin Oksitselanim Septigen Centicin Gentamycins

DTXSID5034642

7440-47-3

VYZAMTAEIAYCRO-UHFFFAOYSA-N

Chromium

Alpaste RRA 030 Alpaste RRA 050 Chromium element Chromium metal

DTXSID3031022

7440-57-5

PCHJSUWPFVWCPO-UHFFFAOYSA-N

Gold

AGC Micro Britecote Burnish Gold C.I. Pigment Metal 3 Colloidal gold Finesphere Gold W 011 Furuuchi 8560 Gold black Gold element Gold Flake Gold Leaf Keradec Palegold 5550 Perfect Gold Shell Gold Technic 504

DTXSID3064697

15663-27-1

DQLATGHUWYMOKM-UHFFFAOYSA-L

Cisplatin

Cis Platinum, diamminedichloro-, (SP-4-2)- Abiplatin Biocisplatinum Briplatin cis-DDP cis-Diaminedichloroplatinum cis-Diaminedichloroplatinum(II) cis-Diaminodichloroplatinum(II) cis-Diamminedichloroplatinum cis-Diamminedichloroplatinum(II) cis-Dichlorodiamineplatinum(II) cis-Dichlorodiammineplatinum cis-Dichlorodiammineplatinum(II) Cismaplat cis-Platin cisplatine cis-Platine cisplatino cis-Platinous diaminodichloride Cisplatinum cis-Platinum cis-Platinum diaminodichloride cis-Platinum II cis-Platinum(II) diaminodichloride cis-Platinum(II) diamminedichloride cis-Platinumdiamine dichloride cis-Platinumdiammine dichloride Cisplatyl Citoplatino Lederplatin lipoplatin Neoplatin NSC 119875 Platamine Platiblastin Platidiam Platinex Platinol Platinol AQ Platinoxan Platinum, diamminedichloro-, cis- Platistin Platosin SPI 077B103 cis-Dichlorodiamine platinum cis-Dichloro diaminoplatinum II

DTXSID4024983

GO:0072559

GO NLRP3 inflammasome complex

CHEBI:26523

CHEBI reactive oxygen species

FMA:84050

FMA Cytokine

FMA:241981

FMA Chemokine

FMA:86578

FMA Interleukin

PR:000001393

PR interleukin-6

PR:000001136

PR interleukin-1 beta

PR:000000134

PR tumor necrosis factor alpha

CHEBI:82594

CHEBI Ferritin

PR:000005897

PR C-reactive protein

CL:0000542

CL lymphocyte

UBERON:0002113

UBERON kidney

CL:0009002

CL inflammatory cell

CL:0000235

CL macrophage

GO:0005739

GO mitochondrion

CL:0000233

CL platelet

CHEBI:5054

CHEBI Fibrin

D009026

MESH mortality

GO:0044546

GO NLRP3 inflammasome complex assembly

MP:0003674

MP oxidative stress

GO:0034599

GO cellular response to oxidative stress

GO:0000302

GO response to reactive oxygen species

GO:0008219

GO cell death

GO:0002526

GO acute inflammatory response

GO:0002534

GO cytokine production involved in inflammatory response

GO:0090195

GO chemokine secretion

GO:0006956

GO complement activation

GO:0002544

GO chronic inflammatory response

GO:0002676

GO regulation of chronic inflammatory response

GO:0032635

GO interleukin-6 production

GO:0032611

GO interleukin-1 beta production

GO:1990774

GO tumor necrosis factor secretion

HP:0012649

HP Increased inflammatory response

HP:0003281

HP Increased serum ferritin

GO:0004457

GO lactate dehydrogenase activity

HP:0011227

HP Elevated C-reactive protein level

HP:0001888

HP Lymphopenia

GO:0050663

GO cytokine secretion

MP:0003606

MP kidney failure

Q000633

MESH toxicity

GO:0043122

GO regulation of I-kappaB kinase/NF-kappaB signaling

MP:0001860

MP liver inflammation

GO:0006954

GO inflammatory response

GO:0042116

GO macrophage activation

HP:0004936

HP Venous thrombosis

GO:0042730

GO fibrinolysis

GO:0072378

GO blood coagulation, fibrin clot formation

WIKI increased

WIKI decreased

WIKI occurrence

WIKI functional change

cell free mitochondrial DNA (mtDNA)

2021-03-19T10:49:02

2021-03-29T07:07:24

unmethylated CpG motifs (DNA;RNA;ODN)

2021-03-19T11:04:48

2021-03-29T06:15:18

SARS-CoV

2020-03-01T10:42:46

Sars-CoV-2 Virus from the coronaviridae family related to SARS-CoV, 229E, NL63, OC43, HKU1 and MERS.

Transmitted by aerosols

2021-02-23T04:50:40

2022-09-09T05:09:36

Danger Associated Molecular Patters (DAMPs)

2021-03-26T04:13:09

Pathogen Associated Molecular Patterns (PAMPs)

2021-03-26T04:14:19

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

Stressor:624 SARS-CoV-2

2021-04-20T03:40:36

Food deprivation

2021-09-06T07:33:54

Gentamicin

2017-10-25T08:30:15

Chromium

2022-02-03T11:22:01

Gold

2022-02-07T15:25:56

Cisplatin

2022-02-03T11:34:57

Reactive oxygen species

2017-06-16T08:32:10

2017-08-15T10:43:27

Nanoparticles and Micrometer Particles

2022-02-04T13:43:43

WCS_9606

common toxicological species humans

10095

NCBI mice

WikiUser_22

all species

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

WCS_7955

common ecological species zebrafish

10116

NCBI rat

WikiUser_28

Vertebrates

10116

NCBI rats

WikiUser_15

ApacheUser Sprague-Dawley

Prolonged TLR9 activation

TLR9 activation

Molecular

AOPWiki 2021-03-19T09:48:14

2021-03-26T06:53:26

Multi Organ Failure involving Acute Respiratory Distress Syndrome

ARDS and MODS

Individual

This KE is created as part of the CIAO-project to describe KEs and AO related to TLR dysregulation (AOP377) and to describe severe outcomes of SARS-CoV-2 in which Acute respiratory distress syndrome (ARDS) is a prominent manifestations of the Multiple organ dysfunction syndrome (MODS), multi-organ failure and a common cause of death for patients admitted to intensive care unit (ICU).

AOPWiki 2021-03-19T09:49:28

2021-11-19T05:40:22

systemic inflammatory response syndrome

SIRS

Individual

AOPWiki 2021-03-19T09:50:36

2021-03-19T09:50:36

Toll Like Receptor (TLR) Dysregulation

TLR Activation/Dysregulation

Molecular

Background Toll-like receptors (TLRs) are a family of 13 conserved transmembrane receptors that are at the forefront of directing innate and adaptive immune responses against invading bacteria, fungi, viruses and parasites (Akira 2003, Takeda, Akira 2004, Pasare, Medzhitov 2005, Tal, Adini et al. 2020, van der Made, Simons et al. 2020). Upon activation TLRs initiate overlapping and distinct signaling pathways in various cell types such as macrophages (MP), conventinal DC (cDC), plasmacytoid DC (pDC), lamina propria DC (LPDC), and inflammatory monocytes (iMO). Engagement of TLR with specific stressors (e.g. PAMPs and DAMPs) induces conformational changes of TLRs that allow homo- or heterophilic interactions of TLRs and recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM to control intracellular signalling pathways leading to the synthesis and secretion of appropriate cytokines and chemokines by cells of the immune system. TLRs have various biological roles both in pathogen combat and tissue homeostasis. This KE is first developed in context of COVID-19 CIAO project. The key gatekeepers in detecting and combating viral infections are TLR3, TLR7, TLR8 and TLR9 and these are predominantly localized in intracellular compartments. In the setting of COVID-19, multiple TLRs are likely relevant in viral combat. Literature covering TLR triggering via SARS-CoV-2 derived PAMPS (Pathogen Associated Molecular Patterns) include: <ul style="margin-left:40px"> <li>TLR7 and TLR8 (+TLR3, TLR4, TLR6)  (Khanmohammadi and Rezaei, 2021)</li> <li>TLR1, TLR4 and TLR6 activated by SARS-CoV-2 spike proteins (Choudhury A et al, 2020)</li> <li>TLR9: Less CpG suppression in coronavirus compared to other viruses, for SARS-CoV-2 in the Envelope (E) open reading frame (E-ORF) and ORF10 (Ng et al., 2004; Digard et al. 2020) and multidisciplinary links described in Bezemer and Garssen, 2021</li> </ul> TLR dysregulation can be multi-fold: <ol> <li>Underperformance of TLR function leading to poor pathogen combat. This is covered in AOP 378</li> </ol> <ul style="margin-left:40px"> <li>COVID-19 patients having poor TLR function (due to polymorphisms) could potentially have less viral clearance capability and more adverse events leading to more severe disease and mortality. This has been shown for TLR7 loss of function polymorphisms (van der Made et al 2020). Knowledge Gap: it is not known if loss of function of other TLRs has a worse outcome in COVID-19 patients.</li> </ul> <ol start="2"> <li>Overperformance of TLR function contributing to exaggerated immune response/cytokine storm/thrombosis/progression into ARDS and MOD. This is covered in AOP377</li> </ol> <ul style="margin-left:40px"> <li>TLR7 and TLR9 expression, measured by RNAseq gene analysis, is more elevated in black Americans than white Americans, which is proposed to explain in part the racial disparity in Covid-19 mortality rates via TLR mediated DC activation (Tal et al. 2020)</li> <li>genetic mutations leading to TLR9 gain of function in human is associated with immune-mediated disease and with a higher incidence of ICU acquired infection (Chatzietal.,2018;Ng et al.,2010).</li> <li>Higher presence of host derived TLR stressors in vulnerable patients can contribute to TLR overstimulation/dysregulation. (Bezemer and Garssen, 2021)</li> </ul> Different classes of "stressors" act on TLR activation/dysregulation <ul style="margin-left:40px"> </ul> 1.  Pathogen associated molecular patterns (PAMPs). TLRs can sense PAMPS during infection or upon exposure to stressors containing micro-organisms or fragments thereof (e.g. cigarette smoke, bioaerosols, house dust mite) <ul style="margin-left:40px"> <li>TLR1 is activated by bacterial Lipopeptides</li> <li>TLR2 is activated by bacterial lipoproteins and glycolipids, TLR2 can form conformations with TLR1 and TLR6 to distinguish between diacyl and triacyl lipopeptides.</li> <li>TLR3 is activated by viral double stranded RNA(dsRNA)</li> <li>TLR4 is activated by Bacterial LPS</li> <li>TLR5 is activated by Bacterial flagellig</li> <li>TLR6 is activated by Bacterial lipopeptides and Fungal zymosan</li> <li>TLR7 and 8 recognize viral single stranded RNA(ssRNA) and bacterial RNA. </li> <li>TLR9 recognizes RNA and DNAmotifs that are rich in unmethylated Cytosine-phosphate-Guanine (CpG) sequences. CpG-motifs are higher expressed in the bacterial and viral genome compared to the vertebrate genome (Hemmi et al., 2000). </li> </ul> 2. host derived Damage-Associated Molecular Patterns (DAMPS). Note that in the context and nomenclature of AOP these DAMPS cannot be labeld as "stressors" since they are derived from inside and not from outside, however these "pseudostressors" do act on the TLR receptors in similar way as PAMPs <ul style="margin-left:40px"> <li> TLR2 and TLR4 are activated by heat shock proteins 60 and 70  (HSP60 and HSP70); extracellular matrix components (ECM); oligosaccharides of hyaluronic acid (HA) and heparan sulfate (HS) (Piccinini AM and Midwood KS, 2010). </li> <li> high-mobility group protein B1 (HMGB1) triggers TLR2, TLR4 and TLR9 </li> <li>Oxidative injury/Oxidized phospholipids  triggers TLR4 mediated NET formation</li> <li>Human mitochondrial DNA (mtDNA), evolutionary derived from endosymbiont bacteria, contains unmethylated CpG-motifs and triggers inflammatory responses directly via TLR9 during injury and/or infection (Zhang et al., 2010).</li> <li>Altered self-ligands, called carboxy-alkyl-pyrroleprotein adducts (CAPs), that are generated during oxidative stress, are known to aggravate TLR9/MyD88 pathway activation (Zhanget al., 2010;Panigrahi et al., 2013). CAPs have been shown to promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo (Panigrahi et al., 2013). </li> </ul> 3. synthetic TLR triggers/blockers (agonists/antagonists) for therapeutic purposes. Examples include CpG-ODNs triggering TLR9 for vaccin adjuvants/cancer treatment/immuno-modulation   Several Modulating factors can contribute to TLR activation/dysregulation <ul style="margin-left:40px"> <li>Co-infection and Trauma (for instance ventilator induced damage) can induce increased levels of TLR9 stressor, mtDNA, which is known to drive worse outcome at ICU in setting of other disorders.  </li> <li>High levels of Visceral Fat, can increase TLR9 expression levels ánd circulating mtDNA</li> <li>Aging triggers both immunosenescence and inflammaging in part via impaired TLR function versus inappropriate triggering via increases of circulating DAMPS (Shaw et al 2011)</li> <li>Genetic polymorphisms can lead to TLR dysregulation (TLR9 gain of function and TLR7 loss of function with worse outcome at ICU Chatzi et al 2018, van der Made et al 2020, Chen et al 2011, )</li> <li>Circulating DAMPS such as mtDNA levels increase with age which is a familiar trait contributing to chronic inflammation, so called“inflamm-aging”in elderly people (Pinti et al., 2014).</li> <li>Vitamin D inhibits expression levels of TLR9</li> <li>Men, higher testosterone, higher TLR4</li> </ul>

Patient specific Ex vivo analysis <ul> <li>Levels of TLR specific stressors (for instance for TLR9, cell free DNA/RNA, mtDNA) are measurable in biological samples (serum, plasma)</li> <li>TLR gain of function and loss of function polymorphisms are measurable</li> <li>TLR expression levels on different cell types and different tissues are measurable by mRNA analysis and by protein analysis</li> <li>TLR function in response to stressors is measurable by analysing components of downstream cascades and read outs of inflammatory mediators (IL6, IL8, IL10, Il17, INF, TNFalpha, etc). This can be done by ex vivo stimulations of cells isolated from patients for instance PBMCs. </li> </ul> In vitro/ in vivo models <ul> <li>TLR Reporter assays</li> <li>TLR knock-out mice</li> </ul>

Cell applicability: TLRs are broadly expressed on various cell types. Examples include: epithelial cells, macrophages, neutrophils, platelets, dendritic cells, NK cells, Tcells, Bcells, neurons, Adipocytes. Tissue/organ level : TLRs are broadly expressed in all vital tissues/organs: lung, heart, liver, spleen, kidney, brain, muscle, gut, skin Taxonomic Applicability: TLRs are well conserved across species but between species variations are reported in terms of sensitivity towards stressors.  For instance certain CpG-ODNs have a stronger TLR9 activating potential in mice than in human and vice versa. Life Stages: TLRs are expressed in all life stages but age variation of level of TLR activation/dysregulation are reported. In elderly immunoscenescence and inflammation are both linked to TLR dysregulation Sex Applicability: Male and female subjects both express functionally active TLRs but sex differences have been reported. For instance certain TLR gain and/or loss of function polymorphisms have higher prevalence in men. Example of TLR7 loss of function (van der Made et al 2020) and TLR9 gain of function (Gao et al 2018, Traub et al 2012, Elsherif et al 2019). Higher testosterone in men has also been linked to higher TLR4 expression. TLR7 is located in a region on the X-chromosome which have a high chance of escaping inactivation leading to higher expression levels in women. Estrogens trigger TLR7, which is higher in women. Exposure of Peripheral blood mononuclear cells (PBMC) to TLR7 ligands will cause a higher production of type I IFN (IFN-a) in female cells than male cells.  (Kovats, 2015;  Takahashi and Iwasaki, 2021; Libert et al.,  2010; Scully et al., 2020)

Moderate Mixed High Male

High

Birth to < 1 month

High

Old Age

High

All life stages

High High High AKIRA, S., 2003. Toll-like receptor signaling. Journal of Biological Chemistry, 278(40), pp. 38105-38108. <div> <div>Gillina F. G. Bezemer, Seil Sagar, Jeroen van Bergenhenegouwen, Niki A. Georgiou, Johan Garssen, Aletta D. Kraneveld and Gert Folkerts</div> </div> <div>Dual role of TLRs in asthma and COPD. Pharmacological Reviews April 1, 2012, 64 (2) 337-358; DOI: https://doi.org/10.1124/pr.111.004622</div> <div> </div> BEZEMER, G.F.G. and GARSSEN, J., 2021. TLR9 and COVID-19: A Multidisciplinary Theory of a Multifaceted Therapeutic Target. Frontiers in pharmacology, 11, pp. 601685. KAWAI, T. and AKIRA, S., 2011. Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity. Immunity, 34(5), pp. 637-650. PASARE, C. and MEDZHITOV, R., 2005. Toll-like receptors: Linking innate and adaptive immunity. Mechanisms of Lymphocyte Activation and Immune Regulation X: Innate Immunity, 560, pp. 11-18. Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signalling. Mediators Inflamm. 2010;2010:672395. doi:10.1155/2010/672395 Shaw AC, Panda A, Joshi SR, Qian F, Allore HG, Montgomery RR. Dysregulation of human Toll-like receptor function in aging. Ageing Res Rev. 2011;10(3):346-353. doi:10.1016/j.arr.2010.10.007 TAKEDA, K. and AKIRA, S., 2004. TLR signaling pathways. Seminars in immunology, 16(1), pp. 3-9. TAL, Y., ADINI, A., ERAN, A. and ADINI, I., 2020. Racial disparity in Covid-19 mortality rates - A plausible explanation. Clinical immunology (Orlando, Fla.), 217, pp. 108481. VAN DER MADE, C.I., SIMONS, A., SCHUURS-HOEIJMAKERS, J., VAN DEN HEUVEL, G., MANTERE, T., KERSTEN, S., VAN DEUREN, R.C., STEEHOUWER, M., VAN REIJMERSDAL, S.V., JAEGER, M., HOFSTE, T., ASTUTI, G., COROMINAS GALBANY, J., VAN DER SCHOOT, V., VAN DER HOEVEN, H., HAGMOLEN OF TEN HAVE, W., KLIJN, E., VAN DEN MEER, C., FIDDELAERS, J., DE MAST, Q., BLEEKER-ROVERS, C.P., JOOSTEN, L.A.B., YNTEMA, H.G., GILISSEN, C., NELEN, M., VAN DER MEER, J.W.M., BRUNNER, H.G., NETEA, M.G., VAN DE VEERDONK, F.L. and HOISCHEN, A., 2020. Presence of Genetic Variants Among Young Men With Severe COVID-19. Jama, . Kovats, Cell Immunol. 2015 April; 294(2): 63–69; Takahashi and Iwasaki, Science. 2021 Jan 22;371(6527):347-348 <div>Libert et al., Nat Rev Immunol. 2010 Aug;10(8):594-604</div> Scully EP, et al. Nat Rev Immunol. 2020. PMID: 32528136   AOPWiki 2021-03-26T03:48:51

2021-11-23T16:54:16

Increased Mortality

Population

Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.

Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause. Depending on the species and the study setup, mortality can be measured: <ul> <li>in the lab by recording mortality during exposure experiments</li> <li>in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species</li> <li>in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.</li> </ul>

All living things are susceptible to mortality.

Moderate Unspecific

High

All life stages

High

AOPWiki 2016-11-29T18:41:24

2022-07-08T07:32:26

increased MyD88 activation

MyD88 activation

Molecular

AOPWiki 2021-07-08T06:04:37

2021-07-08T06:04:37

NLRP3 inflammasome activity, increased

inflammasome activity, increased

Molecular

The NLRP3 inflammasome is a critical component of the innate immune system that mediates Caspase 1 (CASP1) activation. The NLRP3 inflammasome is a multimolecular complex composed of the sensor molecule NLRP3, the adaptor protein PYCARD (commonly called ASC), and pro-caspase 1 (Yang et al, 2019b). In activated inflammasome pro-caspase 1 is cleaved into active CASP1 which subsequently cleaves and thus activates highly pro-inflammatory cytokines interleukin-1B (IL1B) and IL18 leading to increased inflammation (Kelley et al, 2019). NLRP3 inflammasome activation can also induce pyroptosis, an inflammatory form of a cell death (Bergsbaken et al, 2009). Inflammasome activation is associated with COVID-19 disease severity and poor clinical outcome (Rodrigues et al, 2021). NLRP3 inflammasomes can assemble in many cell types, including macrophages, dendritic cells, neutrophils, B cells and T cells, epithelial cells, adipocytes, fibroblasts, astrocytes, cardiomyocytes, hepatocytes, etc (Enni et al, 2020; Ershaid et al, 2019; Wree et al, 2014; Wu et al, 2020; Yang et al, 2019a; Zheng et al, 2020).

In activated inflammasome pro-caspase 1 is cleaved into active CASP1 which then cleaves and thus activates IL1B and IL18. A common method of detection of activated inflammasome is the measurement of secreted IL1B and/or IL18 levels with the enzyme-linked immunosorbent assay (ELISA) using specific antibodies (Martinez et al, 2015; Piancone et al, 2018; Shi et al, 2018; Sun et al, 2017; Yaron et al, 2015). Formation of ASC oligomers reflects inflammasome activation thus ASC oligomers are often used to assess NLRP3 activation. Various methods of ASC oligomer or ASC specks detection is described in a thorough review from Zito and co-authors with references to the studies where the discussed methods are used (Zito et al., 2020). For measuring CASP1 activity as a result of inflammasome assembly and activation, Caspase 1 Fluorescein (FLICA) Assay can be used (Guo et al, 2018; Yaron et al., 2015). FLICA allows active CASP1 enzyme fluorescent labelling that can be analyzed using fluorescence microscopy, fluorescence plate reader or flow cytometry. Furthermore, co-immunoprecipitation assays can be used for specific protein interaction detection (e.g. NLRP3 and ASC/PYCARD) as a result of NLRP3 inflammasome assembly (Zito et al., 2020).

Most of the studies on NLRP3 inflammasome activation are derived from mouse and human tissue experiments. The NLRP3 inflammasome activation and downstream inflammatory response is comprehensively documented in and thus applicable for Homo sapiens (Zito et al, 2020).

Farag NS, Breitinger U, Breitinger HG, El Azizi MA (2020) Viroporins and inflammasomes: A key to understand virus-induced inflammation. Int J Biochem Cell Biol 122: 105738 Guo C, Fu R, Wang S, Huang Y, Li X, Zhou M, Zhao J, Yang N (2018) NLRP3 inflammasome activation contributes to the pathogenesis of rheumatoid arthritis. Clin Exp Immunol 194: 231-243 Martinez GJ, Robertson S, Barraclough J, Xia Q, Mallat Z, Bursill C, Celermajer DS, Patel S (2015) Colchicine Acutely Suppresses Local Cardiac Production of Inflammatory Cytokines in Patients With an Acute Coronary Syndrome. J Am Heart Assoc 4: e002128 Piancone F, Saresella M, Marventano I, La Rosa F, Santangelo MA, Caputo D, Mendozzi L, Rovaris M, Clerici M (2018) Monosodium Urate Crystals Activate the Inflammasome in Primary Progressive Multiple Sclerosis. Front Immunol 9: 983 Shah A (2020) Novel Coronavirus-Induced NLRP3 Inflammasome Activation: A Potential Drug Target in the Treatment of COVID-19. Front Immunol 11: 1021 Shi J, Zhao W, Ying H, Zhang Y, Du J, Chen S, Li J, Shen B (2018) Estradiol inhibits NLRP3 inflammasome in fibroblast-like synoviocytes activated by lipopolysaccharide and adenosine triphosphate. Int J Rheum Dis 21: 2002-2010 Sun X, Hao H, Han Q, Song X, Liu J, Dong L, Han W, Mu Y (2017) Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats. Stem Cell Res Ther 8: 241 Xu H, Chitre SA, Akinyemi IA, Loeb JC, Lednicky JA, McIntosh MT, Bhaduri-McIntosh S (2020) SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway. bioRxiv: 2020.2010.2027.357731 Yaron JR, Gangaraju S, Rao MY, Kong X, Zhang L, Su F, Tian Y, Glenn HL, Meldrum DR (2015) K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells. Cell Death Dis 6: e1954 Zito G, Buscetta M, Cimino M, Dino P, Bucchieri F, Cipollina C (2020) Cellular Models and Assays to Study NLRP3 Inflammasome Biology. Int J Mol Sci 21 AOPWiki 2021-06-25T05:21:29

2021-06-25T08:29:40

Increased, Oxidative Stress

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2016-11-29T18:41:29

2022-02-03T14:20:13

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 (2SH àSS) on neighboring amino acids (Antelmann and 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).  Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016). 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 have its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS are 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</li> <li>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</li> <li>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> <li>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation</li> </ul>   <table border="1" cellpadding="1" cellspacing="1"> <tbody> <tr> <td>Assay Type & Measured Content</td> <td>Description</td> <td>Dose Range Studied</td> <td> Assay Characteristics (Length / Ease of use/Accuracy) </td> </tr> <tr> <td> 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, 10 mM 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)”</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-DA Assay Detection of superoxide production (Thiebault et al., 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</td> <td>**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**</td> <td> </td> <td> </td> </tr> </tbody> </table> Direct Methods of Measurement <table cellspacing="0" class="Table" style="border-collapse:collapse; width:623px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:141px"> Method of Measurement  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:151px"> References  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:255px"> Description  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px"> OECD-Approved Assay </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Chemiluminescence  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Lu, C. et al., 2006;  Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 uminol and lucigenin are commonly used to amplify the signal.  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Spectrophotometry  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Direct or Spin Trapping-Based Electron Paramagnetic Resonance (EPR) Spectroscopy  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Nitroblue Tetrazolium Assay  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Amplex Red Assay  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Dichlorodihydrofluorescein Diacetate (DCFH-DA)  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> HyPer Probe  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Cytochrome c Reduction Assay  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> The cytochrome c reduction assay is used to measure O2•– levels. 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Proton-electron double-resonance imagine (PEDRI) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Glutathione (GSH) depletion  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Biesemann, N. et al., 2018)  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> 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">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>).   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Thiobarbituric acid reactive substances (TBARS)  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Griendling, K. K., et al., 2016) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit.   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"> Protein oxidation (carbonylation) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"> (Azimzadeh et al., 2017; Azimzadeh etal., 2015; Ping et al., 2020) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"> Can be determined with enzyme-linked immunosorbent assay (ELISA) or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">Seahorse XFp Analyzer   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">Leung et al. 2018   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">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 cellspacing="0" class="Table" style="border-collapse:collapse; width:623px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:154px"> Method of Measurement  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:139px"> References  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:256px"> Description  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:75px"> OECD-Approved Assay </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Immunohistochemistry  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:139px"> (Amsen, D., de Visser, K. E., and Town, T., 2009) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:256px"> Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Quantitative polymerase chain reaction (qPCR)  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:139px"> (Forlenza et al., 2012) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:256px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> No </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:154px"> Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:139px"> (Jackson, A. F. et al., 2014) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:256px"> 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 style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:75px"> 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

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(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" style="color:#0563c1; text-decoration:underline">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" style="color:#0563c1; text-decoration:underline">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." style="color:#0563c1; text-decoration:underline">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" style="color:#0563c1; text-decoration:underline">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" style="color:#0563c1; text-decoration:underline">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" style="color:#0563c1; text-decoration:underline">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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3892/ijmm.2019.4188</a> AOPWiki 2017-05-30T13:58:17

2023-03-21T15:16:10

Diminished protective oxidative stress response

Diminished Protective Response to ROS

Cellular

Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Reactive intermediates such as peroxides and free radicals can be very damaging to many parts of cells such as proteins, lipids, and DNA. Severe oxidative stress can trigger apoptosis and necrosis. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19) The cellular defence/defense response to oxidative stress includes induction of detoxifying enzymes and antioxidant enzymes. Nuclear factor-erythroid 2-related factor 2 (Nrf2) binds to the antioxidant response elements (ARE) within the promoter of these enzymes and activates their transcription. Inactive Nrf2 is retained in the cytoplasm by association with an actin-binding protein Keap1. Upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs, and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, cytochrome P450, NAD(P)H quinone oxidoreductase, heme oxygenase, and superoxide dismutase. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19) Nrf2, a master regulator of oxidative stress through enhanced expression of anti-oxidant genes of glutathione and thioredoxin-antioxidant systems, has anti-inflammatory, anti-apoptotic, and antioxidant effects. Dimethyl fumarate (DMF), an activator of Nrf2, can decrease inflammation and reactive oxygen species (ROS) through the inhibition of NF-kappaB by inducing anti-oxidant enzymes (Jackson et al, 2014; Hassan et al, 2020; Timpani et al, 2021). Inactivation of Nrf2 causes diminished protective responses to ROS.

Oxidative stress can be measured as follows: 1. Direct detection of reactive oxygen species (ROS) ROS can be detected by intracellular ROS assay, in vitro ROS/RNS assay. Nitric oxide can be detected in intracellular nitric oxide assay (Ashoka et al, 2020). Hydroxyl, peroxyl, or other ROS can be measured using a fluorescence probe, 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA), at fluorescence detection at 480 nm/530 nm. Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range. ROS can be detected by PEGylated bilirubin-coated iron oxide nanoparticles in whole blood (Lee et al, 2020). 2. Measurement of anti-oxidants The level of catalase, glutathione, or superoxide dismutase can be measured as anti-oxidants. Catalase is an anti-oxidative enzyme that catalyses the resolution of hydrogen peroxide (H2O2) into H2O and O2. The chemiluminescence or fluorescence of HRP catalytic reaction can be detected with residual H2O2 and probes (DHBS+AAP, or ADHP (10-Acetyl-3, 7-dihydroxyphenoxazine)). Anti-oxidant capacity is also one of the oxidative stress markers. Oxygen radical antioxidant capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), total antioxidant capacity (TAC), the cell-based exogenous antioxidant assay can be used for measuring the antioxidant capacity. 3. Detection of damages in protein, lipid, DNA or RNA Oxidation of protein can be measured by the detection of protein carbonyl content (PCC), 3-nitrotyrosine, advanced oxidation protein products, or BPDE protein adduct.  DNA oxidation can be detected with 8-oxo-dG / 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA or HPLC (Chepelev et al, 2015; Valavanidis et al, 2009). Lipid peroxides decompose to form malondialdehyde (MDA) and 4, hydroxynonenal (4-HNE), natural bi-products of lipid peroxidation. Lipid peroxidation can be monitored by thiobarbituric acid (TBA) reactive substances in biological samples. MDA and TBA form MDA-TBA adduct in a 1:2 stoichiometry and detected by colorimetric or fluorometric measurement.

Response to ROS occurs in many cell types and tissues in all life stages and the broad range of mammals.

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific

High

All life stages

High

Ashoka, A.H. et al. (2020), “Recent Advances in Fluorescent Probes for Detection of HOCl and HNO”, ACS omega, 5(4), 1730-1742. https://doi.org/10.1021/acsomega.9b03420. Chepelev, N.L. et al. (2015), “HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues”, J Vis Exp, e52697-e52697, https://doi.org/10.3791/52697. Hassan, S.M. et al. (2020), “The Nrf2 Activator (DMF) and Covid-19: Is there a Possible Role?”, Med Arch, 74(2), 134-138. https://doi.org/10.5455/medarh.2020.74.134-138. 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”, Toxicol Appl Pharmacol, 274, 63-77, https://doi.org/10.1016/j.taap.2013.10.019. Lee, D.Y. et al. (2020), “PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood”, Theranostics, 10(5), 1997-2007. https://doi.org/10.7150/thno.39662. Timpani, C.A, E. Rybalka. (2021), “Calming the (Cytokine) Storm: Dimethyl Fumarate as a Therapeutic Candidate for COVID-19.”, Pharmaceuticals, 14(1), 15. https://doi.org/10.3390/ph14010015. Valavanidis, A. et al. (2009), “8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis”, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 27, 120-39. https://doi.org/10.1080/10590500902885684 AOPWiki 2021-04-20T03:34:23

2023-03-09T20:49:35

Propagation, Oxidative stress

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:48

Th17 cell migration and inflammation induction

Cellular

Psoriasis is known to play a major role in the etiology of T cell dysfunction, especially in over activation of the Th17 pathway, which Th17 cells were associated with Th1 and Th2 (Lisa C. et al. 2007) Th17 cell was identified as a cell population that produces different IL17. Abnormal activation of Toll-like receptors (TLR7, 8 and 9) is also involved in the initiation and maintenance of psoriasis. IMO-3100 (an antagonist of TLR7 and 9) and IMO-8400 (an antagonist of TLR7, 8 and 9) has been shown to reduce psoriasis-like skin lesions induced by intradermal administration of IL-23 on the back of mice (Mayte S-F et al. 2013). Immune cell infiltration in psoriasis lesions is composed of CD3 + Th1cell, Th17 cells and CD11c + dendritic cells (DC) (Chamian F et al 2005). Cytokines such as TNF-α, IFN-γ, IL-17, IL-22, IL-23, IL-12, and IL-1β produced from these cells cause an inflammatory cascade. In particular, the IL-23 / Th17 axis plays an important role, and IL-23h is produce in DC, promotes the differentiation of naive CD4 + T cell progenitor cells into the Th17 phenotype, and stimulates the survival and expansion of the Th17 population  (Harrington LE et al. 2005) (Veldhoen M et al. 2006). IL-17 produced from Th17 cells regulates the expression of defensin, S100 family protein and LL-37. These are innate immune responses in the skin and show higher expression of IL-23 in keratinocytes and dermal tissues of psoriatic lesions than in non-lesions (Liang SC et al. 2006). Overproduction of Th1 and TH17 cytokines is a major cause of psoriasis, and glucocorticoid (GC) regulates epidermal differentiation and acts as a potent anti-inflammatory compound to suppress the pathology of psoriasis. Synthetic glucocorticoids are uses to suppress inflammatory disease  including psoriasis, and induce the glucocorticoid-induced leucine zipper (GILZ), a protein that inhibits major immune cell signaling pathways. CILZ is deficient in lesioned skin of psoriasis patients and shows a negative correlation with the expression of pro-inflammatory cytokines IL-1, IL-23, IL-22, and STAT3. Lisa et al. was identified a T cell-specific role of CILZ that limits Th17 cell formation in vitro in response to the Th17-promoting cytokines IL-1β and IL-23 (Lisa M et al.2019). CILZ has the clinical significance of psoriasis as well as the non-redundant function of controlling pathogenic Th17 responses (Lisa M et al.2019). One of the causes of psoriasis is an increase in pathogenic Th17 cells in people with a genetic predisposition stimulated by the production of Th17 polarized cytokines by bone marrow cells. The antibacterial peptide LL37, which forms a complex with nucleic acids released from cells, is an autoantigen that promotes the activation of cutaneous plasmacytoid dendritic cells and myeloid DCs, and Th17 cells are effector cytokines such as IL-17A. It activates keratinocytes directly through release. Activated keratinocytes proliferate abnormally and release inflammatory mediators and chemokines to amplify the inflammatory response (Boehncke WH et al.2015). <div> </div>

IL17 + cell count measurement                                                                                                  Flow cytometric analysis of psoriasis skin biopsy showed increased IL-17 + and IL-22 + CD4 + T cells, Measurement of IL17 protein levels (in skin and serum)                                                                Increased frequency of IL-17 +, CCR6 +, and CCR4 + T cells. IL-22-producing cells (Th-22 cells) that do not produce IL-17 or IFNγ also increased (Benham et al. 2013).

Ras homolog gene family H (RhoH) is a membrane-bound adapter protein involved in proximal T cell receptor signaling, and spontaneously develops chronic dermatitis that closely resembles human psoriasis in RhoH gene-deficient mice. Ubiquitin protein ligase E3 component N recognition 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels are decreased at the lesion site, and protein levels and DNA binding activity of retinoic acid-related orphan receptors are increased is doing. As a result, T cells differentiated into Th17 cells due to increased production of IL-17 and IL-22. These results indicate that RhoH suppresses the differentiation of naive T cells into effector Th17 cells. RhoH is a gene expressed in blood cells, and when RhoH expression decreases in T cells, Th17 cells increase, IL-22 is produced in large quantities, and the epidermis thickens, leading to the formation of psoriasis pathology. Humans with low RhoH expression may become more severe if they suffer from psoriasis.（Journal of Allergy and Clinical Immunology） The effect of the unique gut flora in psoriasis on the development and reactivity of inflammatory cells on the IL-23 / Th17 axis was analyzed in imiquimod-induced psoriasis model mice. Th17, γδ TCR-bearing lymphocytes in the spleen were measured from sterile (GF) mice, broad-spectrum antibiotic mixture-administered (ATB) mice, and conventional (CV) mice. GF mice and ATB-treated mice had fewer Th17 cells and γδTCR + cells than CV mice. This is thought to be due to the symbiotic bacteria that lack microbiota or changes due to ATB treatment reduce pro-inflammatory T cell response and regulate T cell development. In other words, it is proof that the interaction between the microorganisms of Clostridiales and Elysiperotricales and the host affects the reactivity of Th17 cells and is involved in the etiology of imiquimod-induced skin inflammation. The positive effect of antibiotic regulation of the gut flora on skin severity suggests the involvement of the gut and skin axes and is part of the management of psoriasis patients. (Zizana Z et al. 2016) * Wide-area antibiotic mixture (ATB): A mixture of metronidazole, colistin, streptomycin, and vancomycin.

UBERON:0002405

UBERON immune system

CL:0000451

CL dendritic cell

・Lisa C. Zaba, Irma Cardinale, Patricia Gilleaudeau, Mary Sullivan-Whalen, Mayte Suárez-Fariñas, Judilyn Fuentes-Duculan, Inna Novitskaya, Artemis Khatcherian, Mark J. Bluth, Michelle A. Lowes, James G. Krueger. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses. J. Exp. Med. 2007, 204, 3183-3194. ・Mayte Suarez-Farinas, Robert Arbert, Weiwen Jiang, Francesca S. Ortenzio, Tim Sullivan, James G, Krueger.Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor7,8 and 9 Antagonists in a Model of IL-23-Induced Skin Inflammmation. PLOS ONE, December 2013/Vol 8/Issue 12/e84634 ・Chamian F, Lowes MA, Lin SL, Lee E, Kikuchi T et al. (2005)Alefacept reduces infiltrating T cells, activated dendritic cells, and inflammatory genes in psoriasis vulgaris. Proc Natl Acad Sci U S A 102: 2075-2080. ・Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy  TL et al. (2005) Interleukin 17-producing  CD4+ effector  T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6:1123-1132. ・Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B(2006) novo differentiation of IL-17-producing  T cells. Immunity 24:179-189. ・Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K et al. (2006) Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 203:2271-2279.   ・Lisa M. Paloma Perez. Glucocorticoids and Glucocorticoid-Induced-Leucine-Zipper (GILZ) in Psoriasis:Published online 2019 Sep 13. ・Boehncke WH, Schon MP. Psoriasis. Lancet (2015)386(9997);983-94.10.1016/S0140-6736(14)61909-7 ・Helen Benham, Jane C Goodall, Mihir D Wechalekar, and Dliver Fitzgerald. Th17 and Th22 cells in psoriatic arthritis and psoriasis. Arthritis research & therapy September 2013. ・Journal of Allergy and Clinical Immunology ・Zuzana Zakostelska, Jana Malkova, Kiara Klimesova, Pavel Rossmann, Michaela Hornova, Iva Novosadova, Zuzana Stehlikova, Martin Kostovcik, Tomas Hudcovic, Ranata Stepankova, Katerina Juzlova, Jana Hercogova, Helena Tlaskalova-Hogenova, Miloslav Kverka. Intestinal MicrobiotaPromotes Psoriasis-Like Skin Inflammation by Enhancing Th17 Response. PLOS ONE. 2016;Jul 19. AOPWiki 2019-12-27T04:32:56

2020-11-23T06:59:20

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

2022-07-15T09:46:25

Increase, Cell death

Cellular

Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. micro organisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016). Cell death can be divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death (Bertheloot et al., 2021). Programmed cell death: is a form of cell death in which the dying cell plays an active part in its own demise (Cotter & Al-Rubeai, 1995). Apoptosis At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release: <ul> <li>Intrinsic pathway is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol.</li> <li>Extrinsic pathway is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5.</li> </ul> Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« (Blank & Shiloh, 2007). Necroptosis: This type of regulated cell death, occurs following the activation of the tumor necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals. Pyroptosis is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor. Autophagy: is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019). Anoikis is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form od induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant in vivo (Gilmore, 2005). Cornification: is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others (Eckhart et al., 2013). Non-programmed cell death: occurs accidentally in an unplanned manner. Necrosis is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019).

Assays for Quantitating Cell Death: <ul> <li>Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a) </li> <li>TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013).</li> </ul> Assays for Quantitating Cell Survival           Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b). ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES Detecting Nuclear Condensation: The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in color and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in color and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a). Detection of DNA Fragmentation: Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a). TUNEL technique can also be used: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013). Detecting Phosphatidylserine Exposure: Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e). Detecting Caspase Activity: antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labeled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a). Detecting Mitochondrial Damage: flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining (Crowley, Christensen, & Waterhouse, 2015b).     Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.     Measures of apoptotic cytomorphological alterations:   Apoptotic cells exhibit electron dense nuclei, nuclear fragmentation, intact cell membrane up to the disintegration phase, disorganized cytoplasmic organelles, large clear vacuoles, blebs at cell surface, and apoptotic bodies, which can be visualized with various methods. (Elmore, 2007; Watanabe et al., 2002)  <table border="1"> <tbody> <tr> <td> Method of Measurement  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Transmission electron microscopy (TEM) / Scanning electron microscopy (SEM)/ Fluorescence microscopy  </td> <td> Martinez, Reif, and Pappas, 2010; Watanabe et al., 2002  </td> <td> TEM and SEM can image the cytomorphological alterations caused by apoptosis.  </td> <td> No  </td> </tr> <tr> <td colspan="3"> Stains:  </td> <td>   </td> </tr> <tr> <td> Hematoxylin with eosin  </td> <td> Elmore, 2007   </td> <td> Hematoxylin stains nuclei blue and eosin stains the cytoplasm/extracellular matrix pink, allowing for the visualization of the cytomorphological alterations of cells.  </td> <td> No  </td> </tr> <tr> <td> Toluidine blue or methylene blue  </td> <td> Watanabe et al., 2002  </td> <td> Toluidine blue stains cellular nuclei, and identifies malignant tissue, which has an increased DNA content and a higher nuclear-to-cytoplasmic ratio.  Methylene blue stain applied to a healthy cell sample results in a colorless stain. This is due to the cell's enzymes, which reduce the methylene blue, thereby, reducing its color. Methylene blue stain applied to a dead cell sample turns blue.  </td> <td> No  </td> </tr> <tr> <td> DAPI  </td> <td> Crowley, Marfell, and Waterhouse, 2016  </td> <td> Binds strongly to adenine–thymine-rich regions in the DNA. DAPI can stain live and fixed cells. It passes less efficiently through the membrane in live cells.  </td> <td> Yes  </td> </tr> <tr> <td> Hoescht 33342  </td> <td> Crowley, Marfell, and Waterhouse, 2016  </td> <td> Binds to DNA in live and fixed cells, used to measure DNA condensation.  </td> <td> Yes   </td> </tr> <tr> <td> Acridine Orange (AO)  </td> <td> Watanabe et al., 2002  </td> <td> Interacts with DNA/RNA through intercalation/electrostatic interaction, is able to penetrate cell membranes. Stains live cells green and dead cells red.  </td> <td> No  </td> </tr> <tr> <td> Nile blue sulfate  </td> <td> Watanabe et al., 2002  </td> <td> Stains cell nuclei and lysosomes, indicating apoptotic bodies.  </td> <td> No  </td> </tr> <tr> <td> Neutral red  </td> <td> Watanabe et al., 2002  </td> <td> Measures lysosomal membrane integrity  </td> <td> No  </td> </tr> <tr> <td> LysoTracker Red  </td> <td> Watanabe et al., 2002  </td> <td> Measures phagolysosomal activity that occurs due to the engulfment of apoptotic bodies.  </td> <td> No   </td> </tr> </tbody> </table>   DNA damage/fragmentation assays:  <table border="1"> <tbody> <tr> <td> Assay  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay  </td> <td> Kressel and Groscurth, 1994  </td> <td> Apoptosis is detected with the TUNEL method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks.  </td> <td> Yes    </td> </tr> <tr> <td> Nicoletti Assay (SubG1 cell fragment measurement)  </td> <td> Nicoletti et al., 1991  </td> <td> Measures DNA content in nuclei at the pre-G1 phase of the cell cycle (apoptotic nuclei have less DNA than nuclei in healthy cells).  </td> <td> No  </td> </tr> <tr> <td> Cell Death Detection ELISA kit  </td> <td> Parajuli, 2014  </td> <td> Apoptotic nucleosomes are detected using the Cell Death Detection ELISA kit, which were calculated as absorbance subtraction at 405 nm and 490 nm.  </td> <td> No  </td> </tr> </tbody> </table>   Measurement of apoptotic markers through immunochemistry:  <table border="1"> <tbody> <tr> <td> Method of Measurement  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Western blot / immunofluorescence microscopy / immunohistochemistry  </td> <td> Elmore 2007; Martinez, Reif, and Pappas, 2010; Parajuli et al, 2014  </td> <td> Apoptosis can be detected with the expression of various apoptotic markers by western blotting using antibodies. Markers can include: cytosolic cytochrome-c; caspases 2, 3, 6, 7, 8, 9, 10; Bax; Bcl-2 (apoptosis inhibitor); BIRC2; BIRC3; GAPDH; PARP; CDK2; CDK4; cyclin D1; p53; p63; p73; cytokeratin-18  </td> <td> No   </td> </tr> </tbody> </table>   Measures of altered caspase activity:  <table border="1"> <tbody> <tr> <td> Method of Measurement  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm  </td> <td>  Wu, 2016  </td> <td> Visualizes caspase-3 and caspase-9 activity  </td> <td> No  </td> </tr> <tr> <td> PhiPhiLux Assay  </td> <td> Watanabe et al., 2002  </td> <td> The PhiPhiLux molecule becomes fluorescent once it is cleaved by caspase-3, indicating caspase activity.  </td> <td> No  </td> </tr> <tr> <td> Ferrocene reporter  </td> <td> Martinez, Reif, and Pappas, 2010  </td> <td> An electrochemical method to detect apoptosis. Ferrocene is attached to a peptide. The peptide sequence is a caspase 3 cleavage site and the ferrocene acts as the electrochemical reporter. The more caspase cleavage that occurs, the more ferrocene molecules are cleaved, the stronger the signal.  </td> <td> No  </td> </tr> <tr> <td> Self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry (SAMDI-MS) assay  </td> <td> Martinez, Reif, and Pappas, 2010  </td> <td> This assay detects caspase activity.  </td> <td> No  </td> </tr> </tbody> </table>   Measures of altered mitochondrial physiology:  <table border="1"> <tbody> <tr> <td> Method of Measurement  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Laser scanning confocal microscopy (LSCM)  </td> <td> Watanabe et al., 2002  </td> <td> LCSM can monitor many mitochondrial events following staining of cells, such as: mitochondrial permeability transition, depolarization of the inner mitochondrial membrane, which may be indicative of apoptosis.  </td> <td> No    </td> </tr> <tr> <td> Fluorescent, cationic, lipophilic mitochondrial dyes, such as: JC-1 dye, Rhodamine, DiOC6, Mitotracker red  </td> <td> Martinez, Reif, and Pappas, 2010; Sivandzade, Bhalerao, and Cucullo, 2019  </td> <td> These mitochondrial dyes can indicate disintegration of the mitochondrial outer membrane’s electrochemical gradient, as different fluorescence is observed between healthy and apoptotic cells. In healthy cells the dye accumulates in aggregates, but in apoptotic cells missing the electrochemical membrane, the dye will spread out into the cytoplasm providing different fluorescent signals.  </td> <td> No  </td> </tr> </tbody> </table>   Other measures:  <table border="1"> <tbody> <tr> <td> Method of measurement  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Apoptosis PCR microarray  </td> <td> Elmore, 2007  </td> <td> A method to profile the gene expression of many apoptotic-related genes, for example: ligands, receptors, intracellular modulators, and transcription factors.  </td> <td> No  </td> </tr> <tr> <td> Fluorescence correlation spectroscopy (FCS) or dual-colour fluorescence cross-correlation spectroscopy (dcFCCS)   </td> <td> Martinez, Reif, and Pappas, 2010  </td> <td> Used to measure protease activity.  </td> <td> No  </td> </tr> <tr> <td> Apoptosis is measured with Annexin V-FITC probes  </td> <td> Elmore, 2007; Wu et al., 2016  </td> <td> A measure of apoptotic membrane alterations. Annexin-V detects externalized phosphatidylserine residues, a result of apoptosis. Can be conducted in conjunction with propidium iodide (PI) staining. The relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry.  </td> <td> Yes    </td> </tr> </tbody> </table>

The process of cell death is highly conserved within multi‐cellular organisms. (Lockshin & Zakeri, 2004).   Taxonomic applicability: Increased cell death is applicable to all animals. This includes vertebrates such as humans, mice and rats (Alberts et al., 2002).   Life stage applicability: There is insufficient data on life stage applicability of this KE.  Sex applicability: This key event is not sex specific (Forger and de Vries, 2010; Ortona Matarrese, and Malorni, 2014).   Evidence for perturbation by a stressor: Multiple studies show that cell death can be increased or disrupted by many types of stressors including ionizing radiation and altered gravity (Zhu et al., 2016).

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific

High

All life stages

High High High High Alberts, B. et al. (2002), “Programmed Cell Death (Apoptosis)”, in Molecular Biology of the Cell. 4th edition, Garland Science, New York, https://www.ncbi.nlm.nih.gov/books/NBK26873/   Bertheloot, D., Latz, E., & Franklin, B. S. (2021). Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cellular & Molecular Immunology, 18, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3 Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. Journal of Neurocytology, 28(10–11), 781–793. https://doi.org/10.1023/a:1007005702187 Blank, M., & Shiloh, Y. (2007). Cell Cycle Programs for Cell Death: Apoptosis is Only One Way to Go. Cell Cycle, 6(6), 686–695. https://doi.org/10.4161/cc.6.6.3990 Cotter, T. G., & Al-Rubeai, M. (1995). Cell death (apoptosis) in cell culture systems. Trends in Biotechnology, 13(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X Crowley, L. 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2023-03-22T11:07:45

increased release of danger/damage associated molecular patterns

increased release of DAMPS

Tissue

AOPWiki 2021-07-08T07:14:44

2021-07-08T07:14:44

Platelet-neutrophil interactions, Increased

Cellular

<big>Neutrophils represent 50–70% of all circulating leukocytes in humans and are the foot soldiers of the immune response, being the first immune cell on the scene of an infection or inflammation. They exert their functional roles via a combination of processes, including the generation of reactive oxygen species (ROS), degranulation, phagocytosis, and release of neutrophil extracellular traps (NETs).  On the other hand, approximately 750 billion platelets circulate in humans and these cells are traditionally known for their role in haemostasis and thrombosis and are first responders at sites of vascular damage. More recently investigations have shown that platelets play important roles in inflammatory and immune processes (Gu et al., 2019), including resolution (Senchenkova et al., 2019). Platelets have a range of different receptors including Toll-like receptors (TLRs), C-type lectin receptors, and nucleotide-binding and oligomerization domain–like receptors, which are known to recognise infection (Semple et al., 2011) and are effectors of injury in a variety of pulmonary disorders and syndromes, and facilitate tissue repair (Xu et al., 2016).</big> <big>These two cell types modulate each other’s functions, and neutrophil-platelet interaction and platelet-platelet interactions are increased during inflammation (Lisman, 2018). Circulating platelet–neutrophil complexes have been observed in many different inflammatory conditions including bacterial infections (Gawaz et al. 1995) and pulmonary syndromes involving inflammation (Caudrillier et al. 2012). The physical interaction between neutrophils and platelets is mediated by multiple molecular mechanisms including platelet P-selectin binding to neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) (Moore et al. 1995) and platelet glycoprotein Ibα binding to neutrophil MAC-1 (Simon et al. 2000).</big> <big>A first important example of functional interaction between the two cell types is the ability of platelets to facilitate neutrophil adhesion to activated endothelial cells (i.e. at the site of inflammation or injury) and to promote their transmigration across the endothelium. Many studies have demonstrated that neutrophils patrol the vasculature for activated platelets to initiate inflammatory responses (Sreeramkumar et al. 2014), hence activated platelets are crucial in neutrophil-mediated inflammatory responses (Senchenkova et al., 2019). The depletion of platelets decreases neutrophil recruitment to the site of inflammation, and the depletion of neutrophils decreases platelet recruitment (Sreeramkumar et al. 2014). During activation, both neutrophils and platelets are capable of releasing microparticles, (MPs, also termed ectosomes) which carry markers of the parent cells outer membrane e.g. neutrophil MPs carry different cell adhesion molecules and proteases such as proteinase 3 or elastase (Ramirez et al., 2019).</big> <big>Beyond recruitment and localization processes, activated platelets are able to modulate (i.e. both enhancing and inhibiting) neutrophil responses, including phagocytosis, production of ROS, and production of NETs. These effects can be mediated by direct cell-cell contact or by the release of soluble mediators such as CCL5 and platelet factor 4 (von Hundelshausen et al. 2005). On the other hand, neutrophils can also release soluble mediators (e.g. cathepsin G, elastase) that enhance or inhibit platelet responses (Mihara et al. 2013; Bonnefoy and Legrand 2000).</big> <big>Platelets can stimulate the synthesis of various leukotrienes by neutrophils via transcellular transfer of the arachidonic acid metabolite 12-HETE, which is further processed by neutrophils into bioactive leukotrienes (Rossaint et al., 2018). Platelets also store preformed and immunomodulatory molecules that can affect neutrophils (e.g. affecting chemotaxis) and the immune response, including interleukin-1 and platelet-derived growth factor (Semple et al., 2011).</big> <big>Over the past several years, the interactions between immune cells such as neutrophils and platelets have been given different names, including: thromboinflammatoin, immunothrombosis and immunohemostasis (Guo et al., 2019). These names have been introduced to try to capture the responses and mechanisms of both cell types (independently and collectively) in thrombosis and inflammation.</big> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> </tbody> </table>

<big>Neutrophil-platelet complexes (sometimes called platelet-leukocyte aggregation, PLA) can be measured using the following techniques (see <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178087/" style="color:blue; text-decoration:underline">Finsterbusch et al. (2018)</a> for a comprehensive review):</big> <big>In vitro </big> <ol> <li><big>Microfluidic assays can allow to visualize neutrophil-platelet interactions in real time in vitro. This approach involves the use of isolated cells or whole blood that are perfused through a chamber coated with immobilized proteins or cells. Specific cells are visualized thanks to the use of specific antibodies. This technique allows a fine tuning of experimental conditions and the use of human cells. </big></li> </ol> <big>In vivo </big> <ol start="2"> <li><big>Flow cytometry is the method of choice to measure circulating neutrophil-platelet complexes in both humans and animals. This technique is based on fluorescence-labelling of platelets and leukocytes in whole blood. Characteristic side scatter properties of leukocyte subtypes already permit basic discrimination of platelets binding to neutrophils, monocytes and lymphocytes. However, leukocyte subtype-specific antibodies generate more accurate data. The endpoint is quantified as the percentage of total leukocytes (i.e. neutrophils) that also stain for a platelet-specific marker. A limitation of standard flow cytometry is that potential platelet-derived macrovesicles fused to leukocytes could influence results. </big></li> <li><big>Flow cytometry coupled with fluorescence microscopy (imaging flow cytometry). This approach enables direct assessment of both number of bound platelets as well as involved interaction molecules and coincidental events.</big></li> <li><big>Histochemical and immunofluorescent imaging of frozen or paraffin-embedded tissue sections. This approach can provide valuable information about the location of platelet recruitment and neutrophil-platelet complexes within tissue microenvironments of most organs. These techniques can be coupled with confocal or electron microscopy to provide higher resolution visualization of platelet-neutrophil interactions. A major limitation of this approach is that it only provides a static assessment of the endpoint. </big></li> <li><big>Live cell imaging coupled with intravital microscopy (IVM) allows to track labeled cells in live animals over longer time periods in organs including liver, lung, and brain. This approach can be used to analyze the dynamic interactions between platelets and neutrophils (e.g., duration of interactions and behavioural changes in neutrophils and/or platelets upon contact).</big></li> <li><big>Intravenous administration of fluorochrome-conjugated monoclonal antibodies targeting specific protein markers on platelets.</big></li> </ol>

High Bonnefoy, A., Legrand, C., 2000. Proteolysis of Subendothelial Adhesive Glycoproteins (Fibronectin, Thrombospondin, and von Willebrand Factor) by Plasmin, Leukocyte Cathepsin G, and Elastase. Thrombosis Research 98, 323-332. Caudrillier, A., Kessenbrock, K., Gilliss, B.M., Nguyen, J.X., Marques, M.B., Monestier, M., Toy, P., Werb, Z., Looney, M.R., 2012. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest 122, 2661-2671. Finsterbusch, M., Schrottmaier, W.C., Kral-Pointner, J.B., Salzmann, M., Assinger, A., 2018. Measuring and interpreting platelet-leukocyte aggregates. Platelets 29, 677-685. Gawaz, M., Fateh‐Moghadam, S., Pilz, G., Gurland, H.-., Werdan, K., 1995. Platelet activation and interaction with leucocytes in patients with sepsis or multiple organ failure. European Journal of Clinical Investigation 25, 843-851. Guo, L., Rondina, M.T., 2019. The Era of Thromboinflammation: Platelets Are Dynamic Sensors and Effector Cells During Infectious Diseases. Front Immunol 10. Lisman, T., 2018. Platelet–neutrophil interactions as drivers of inflammatory and thrombotic disease. Cell Tissue Res 371, 567-576. Mihara, K., Ramachandran, R., Renaux, B., Saifeddine, M., Hollenberg, M.D., 2013. Neutrophil Elastase and Proteinase-3 Trigger G Protein-biased Signaling through Proteinase-activated Receptor-1 (PAR1). J Biol Chem 288, 32979-32990. Moore, K.L., Patel, K.D., Bruehl, R.E., Li, F., Johnson, D.A., Lichenstein, H.S., Cummings, R.D., Bainton, D.F., McEver, R.P., 1995. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. Journal of Cell Biology 128, 661-671. Ramirez, G.A., Manfredi, A.A., Maugeri, N., 2019. Misunderstandings Between Platelets and Neutrophils Build in Chronic Inflammation. Front Immunol 10. Rossaint, J., Margraf, A., Zarbock, A., 2018. Role of Platelets in Leukocyte Recruitment and Resolution of Inflammation. Front Immunol 9. Semple, J.W., Italiano, J.E., Freedman, J., 2011. Platelets and the immune continuum. Nature Reviews Immunology 11, 264-274. Senchenkova, E.Y., Ansari, J., Becker, F., Vital, S.A., Al-Yafeai, Z., Sparkenbaugh, E.M., Pawlinski, R., Stokes, K.Y., Carroll, J.L., Dragoi, A., Qin, C.X., Ritchie, R.H., Sun, H., Cuellar-Saenz, H.H., Rubinstein, M.R., Han, Y.W., Orr, A.W., Perretti, M., Granger, D.N., Gavins, F.N.E., 2019. Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation. Circulation 140, 319-335. Simon, D.I., Chen, Z., Xu, H., Li, C.Q., Dong, J., McIntire, L.V., Ballantyne, C.M., Zhang, L., Furman, M.I., Berndt, M.C., López, J.A., 2000. Platelet Glycoprotein Ibα Is a Counterreceptor for the Leukocyte Integrin Mac-1 (Cd11b/Cd18). J Exp Med 192, 193-204. Sreeramkumar, V., Adrover, J.M., Ballesteros, I., Cuartero, M.I., Rossaint, J., Bilbao, I., Nácher, M., Pitaval, C., Radovanovic, I., Fukui, Y., McEver, R.P., Filippi, M., Lizasoain, I., Ruiz-Cabello, J., Zarbock, A., Moro, M.A., Hidalgo, A., 2014. Neutrophils scan for activated platelets to initiate inflammation. Science 346, 1234-1238. von Hundelshausen, P., Koenen, R.R., Sack, M., Mause, S.F., Adriaens, W., Proudfoot, A.E.I., Hackeng, T.M., Weber, C., 2005. Heterophilic interactions of platelet factor 4 and RANTES promote monocyte arrest on endothelium. Blood 105, 924-930. Xu, X.R., Zhang, D., Oswald, B.E., Carrim, N., Wang, X., Hou, Y., Zhang, Q., Lavalle, C., McKeown, T., Marshall, A.H., Ni, H., 2016. Platelets are versatile cells: New discoveries in hemostasis, thrombosis, immune responses, tumor metastasis and beyond. Critical Reviews in Clinical Laboratory Sciences 53, 409-430. AOPWiki 2021-04-08T07:53:35

2021-04-08T08:49:34

Increased Pro-inflammatory mediators

Increased pro-inflammatory mediators

Tissue

Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators. Exogenous mediators of inflammation are bacterial products or toxins like endotoxin or LPS. Endogenous mediators of inflammation are produced from within the (innate and adaptive) immune system itself, as well as other systems. They can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation, and kinin systems. Or they can be released at the site of injury by a number of cell types that either contain them as preformed molecules within storage granules, e.g. histamine, or which can rapidly switch on the machinery required to synthesize the mediators. Table1: a non-exhaustive list of examples for pro-inflammatory mediators <table border="1" cellpadding="0" cellspacing="0"> <tbody> <tr> <td style="width:253px"> Classes of inflammatory mediators </td> <td style="width:361px"> Examples </td> </tr> <tr> <td style="width:253px"> Pro-inflammatory cytokines </td> <td style="width:361px"> TNF-a, Interleukins (IL-1, IL-6, IL-8), Interferons  (IFN-g), chemokines (CXCL, CCL, GRO-α, MCP-1), GM-CSF </td> </tr> <tr> <td style="width:253px"> Prostaglandins </td> <td style="width:361px"> PGE2 </td> </tr> <tr> <td style="width:253px"> Bradykinin </td> <td style="width:361px">   </td> </tr> <tr> <td style="width:253px"> Vasoactive amines </td> <td style="width:361px"> histamine, serotonin </td> </tr> <tr> <td style="width:253px"> Reactive oxygen species (ROS) </td> <td style="width:361px"> O2-, H2O2 </td> </tr> <tr> <td style="width:253px"> Reactive nitrogen species (RNS) </td> <td style="width:361px"> NO, iNOS </td> </tr> </tbody> </table> The increased production of pro-inflammatory mediators can have negative consequences on the parenchymal cells leading even to cell death, as described for TNF-a or peroxynitrite on neurons (Chao et al., 1995; Brown and Bal-Price, 2003). Along with TNF-α, IL-1β and IL-6 have been shown to exhibit negative consequences on neurogenesis and neuronal precursor cell proliferation when overexpressed. IFN-γ is also associated with neuronal damage, although it is not as extensively studied compared to TNF-α, IL-1β and IL-6. In addition, via a feedback loop, they can act on the reactive resident cells thus maintaining or exacerbating their reactive state; and by modifying elements of their signalling pathways, they can favour the M1 phenotypic polarization and the chronicity of the inflammatory process (Taetzsch et al., 2015). Basically, this event occurs equally in various tissues and does not require tissue-specific descriptions. Nevertheless, there are some specificities such as the release of glutamate by brain reactive glial cells (Brown and Bal-Price, 2003; Vesce et al., 2007).The differences may rather reside in the type of insult favouring the increased expression and/or release of a specific class of inflammatory mediators, as well the time after the insult reflecting different stages of the inflammatory process. For these reasons, the analyses of the changes of a battery of inflammatory mediators rather than of a single one is a more adequate measurement of this KE. Regulatory examples using the KE CD54 and CD 86 as well as IL-8 expression is used to assess skin sensitization potential (OECD TG 442E). IL-2 expression is used to assess immunotoxicity (and will become an OECD test guideline); for the latter see also doi: 10.1007/s00204-018-2199-7.   LIVER: When activated, resident macrophages (Kupffer cells) release inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and are a main source of TGF-β1 - the most potent pro-fibrogenic cytokine. Following the role of TGF-β is described in more detail. Transforming growth factor β (TGF-β) is a pleiotropic cytokine with potent regulatory and inflammatory activity [Sanjabi et al., 2009; Li and Flavell, 2008a;2008b]. The multi-faceted effects of TGF-β on numerous immune functions are cellular and environmental context dependent [Li et al., 2006]. TGF-β binds to TGF-β receptor II (TGF-βRII) triggering the kinase activity of the cytoplasmic domain that in turn activates TGF-βRI. The activated receptor complex leads to nuclear translocation of Smad molecules, and transcription of target genes [Li et al., 2006a]. The role of TGF-β as an immune modulator of T cell activity is best exemplified by the similarities between TGF-β1 knockout and T cell specific TGF-β receptor II knockout mice [Li et al., 2006b; Marie et al., 2006;Shull et al., 1992]. The animals in both of these models develop severe multi-organ autoimmunity and succumb to death within a few weeks after birth [Li et al., 2006b; Marie et al., 2006; Shull et al., 1992]. In addition, in mice where TGF-β signaling is blocked specifically in T cells, the development of natural killer T (NKT) cells, natural regulatory T (nTreg) cells, and CD8+ T cells was shown to be dependent on TGF-β signaling in the thymus [Li et al., 2006b; Marie et al., 2006]. TGF-β plays a major role under inflammatory conditions. TGF-β in the presence of IL-6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [Korn et al., 2009]. TGF-β orchestrates the differentiation of both Treg and Th17 cells in a concentration-dependent manner [Korn et al., 2008]. In addition, TGF-β in combination with IL-4, promotes the differentiation of IL-9- and IL-10-producing T cells, which lack suppressive function and also promote tissue inflammation [Dardalhon  et al., 2008; Veldhoen et al., 2008]. The biological effects of TGF-β under inflammatory conditions on effector and memory CD8+ T cells are much less understood. In a recent study, it was shown that TGF-β has a drastically opposing role on naïve compared to antigen-experienced/memory CD8+ T cells [Filippi et al., 2008]. When cultured in vitro, TGF-β suppressed naïve CD8+ T cell activation and IFN-γ production, whereas TGF-β enhanced survival of memory CD8+ T cells and increased the production of IL-17 and IFN-γ [Filippi et al., 2008]. TGF-β also plays an important role in suppressing the cells of the innate immune system. The transforming growth factor beta (TGF-β) family of cytokines are ubiquitous, multifunctional, and essential to survival. They play important roles in growth and development, inflammation and repair, and host immunity. The mammalian TGF-β isoforms (TGF-β1, β2 and β3) are secreted as latent precursors and have multiple cell surface receptors of which at least two mediate signal transduction. Autocrine and paracrine effects of TGF-βs can be modified by extracellular matrix, neighbouring cells and other cytokines. The vital role of the TGF-β family is illustrated by the fact that approximately 50% of TGF-1 gene knockout mice die in utero and the remainder succumb to uncontrolled inflammation after birth. The role of TGF-β in homeostatic and pathogenic processes suggests numerous applications in the diagnosis and treatment of various diseases characterised by inflammation and fibrosis. [Clark and Coker, 1998; Santibañez et al., 2011; Pohlers et al., 2009] Abnormal TGF-β regulation and function are implicated in a growing number of fibrotic and inflammatory pathologies, including pulmonary fibrosis, liver cirrhosis, glomerulonephritis and diabetic nephropathy, congestive heart failure, rheumatoid arthritis, Marfan syndrome, hypertrophic scars, systemic sclerosis, myocarditis, and Crohn’s disease. [Gordon and Globe,2008] TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines. TGF-β is synthesized as a non-active pro-form, forms a complex with two latent associated proteins latency-associated protein (LAP) and latent TGF- β binding protein (LTBP) and undergoes protolithic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Among the TGF-βs, six distinct isoforms have been discovered although only the TGF-β1, TGF-β2 and TGF-β3 isoforms are expressed in mammals, and their human genes are located on chromosomes 19q13, 1q41 and 14q24, respectively. Out of the three TGF-β isoforms (β1, β2 and β3) only TGF-β1 was linked to fibrogenesis and is the most potent fibrogenic factor for hepatic stellate cells. [Roberts, 1998; Govinden and Bhoola, 2003]. During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TGF-β receptors [Qi et al.; 1999; Shek and Benyon , 2004; De Gouville et al., 2005; Chen et al., 2009]. TGF-β1 induces its own mRNA to sustain high levels in local sites of injury. The effects of TGF-β1 are classically mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. [Parsons et al., 2013; Friedman, 2008; Kubiczkova et al., 2012] Smad1/5/8, MAP kinase (mitogen-activated protein) and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects. TGF-β is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-β is produced by many, but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-β. In general, the release and activation of TGF-β stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins. [Branton and Kopp, 1999] TGF-β 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. [Letterio and Roberts; 1998] In the liver TGF-β1 is released by activated Kupffer cells, liver sinusoidal endothelial cells, and platelets; in the further course of events also activated hepatic stellate cells express TGF-β1. Hepatocytes do not produce TGF-β1 but are implicated in intracellular activation of latent TGF-β1. [Roth et al., 1998; Kisseleva and Brenner, 2007; Kisseleva and Brenner, 2008; Poli, 2000; Liu et al., 2006] TGF-β1 is the most established mediator and regulator of epithelial-mesenchymal-transition (EMT) which further contributes to the production of extracellular matrix. It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4. [Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman,2007; Brenner,2009; Kaimori et al., 2007; Gressner et al., 2002; Kershenobich Stalnikowitz and Weisssbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 200; Parsons et al., 2007; Friedman 2008; Liu et al., 2006] TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the activation of vascular endothelial growth factor (VEGF) High levels of VEGF and TGF-β1 are present in many tumors. Crosstalk between the signalling pathways activated by these growth factors controls endothelial cell apoptosis and angiogenesis. [Clark and Coker; 1998]

The specific type of measurement(s) might vary with tissue, environment and context and will need to be described for different tissue contexts  as used within different AOP descriptions. In general, quantification of inflammatory markers can be done by: <ul> <li>qRT-PCR (mRNA expression)</li> <li>ELISA</li> <li>Immunocytochemistry</li> <li>Immunoblotting</li> </ul> For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al.,  2014   LIVER: There are several assays for TGB-β1 measurement available. e.g. Human TGF-β1 ELISA Kit. The Human TGF-β 1 ELISA (Enzyme –Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-β1 in serum, plasma, cell culture supernatants, and urine. This assay employs an antibody specific for human TGF-β1 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-β1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-β1 antibody is added. After washing away unbound biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of TGF-β1 bound. The StopSolution changes the colour from blue to yellow, and the intensity of the colour is measured at 450 nm [Mazzieri et al., 2000] Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed. <table border="1"> <tbody> <tr> <td> Assay  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> <ul> <li> RT-qPCR  </li> <li> Q-PCR  </li> </ul> </td> <td> (Veremeyko et al., 2012; Alwine et al, 1977; Forlenza et al., 2012)  </td> <td> Measures mRNA expression of cytokines, chemokines and inflammatory markers   </td> <td> No  </td> </tr> <tr> <td> Immunoblotting (western blotting)  </td> <td> (Lee et al., 2008)  </td> <td> Uses antibodies specific to proteins of interest, can used to detect presence of pro-inflammatory mediators in samples of cell or tissue lysate  </td> <td> No  </td> </tr> <tr> <td> Whole blood stimulation assay  </td> <td> (Thurm & Halsey, 2005)  </td> <td>  Detects inflammatory cytokines in blood  </td> <td> No  </td> </tr> <tr> <td> Imaging tests  </td> <td> (Rollins & Miskolci, 2014)  </td> <td> A qualitative technique using a cytokine specific antibodies and fluorophores can be used to visualize expression patterns, subcellular location of the target and protein-protein interactions.   Common examples include double immunofluorescence confocal microscopy or other molecular imaging modalities.  </td> <td> No  </td> </tr> <tr> <td> Flow-cytometry  </td> <td> (Karanikas et al., 2000)  </td> <td> Detects the intracellular cytokines with stimulation.  </td> <td> No  </td> </tr> <tr> <td> Immunoassays (ex. enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), radioimmunoassay)  </td> <td> (Amsen et al., 2009; Engvall & Perlmann, 1972; Ji & Forsthuber, 2016; Goldsmith, 1975)  </td> <td> Plate based assay technique using antibodies to detect presence of a protein in a liquid sample.   Can be used to identify presence of an inflammatory cytokine of interest especially when in low concentrations.   </td> <td> No  </td> </tr> <tr> <td> Inflammatory cytokine arrays    </td> <td> (Amsen et al., 2009)    </td> <td> Similar to the ELISA, except using a membrane-based rather than plate-based approach. Can be used to measure multiple cytokine targets concurrently.   </td> <td> No  </td> </tr> <tr> <td> Immunohistochemistry (IHC)  </td> <td> (Amsen et al., 2009; Coons et al., 1942)  </td> <td> Immobilized tissue or cell cultures are stained using antibodies for specificity of ligands of interest. Versions of the assays can be used to visualize localization of inflammatory cytokines.   </td> <td> No  </td> </tr> </tbody> </table>

LIVER: Human [Santibañez et al., 2011] Rat [Luckey and Petersen, 2001] Mouse [Nan et al., 2013] BRAIN: Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al.,  2014   Taxonomic applicability: The inflammatory response and increase of the pro-inflammatory mediators has been observed across species from simple invertebrates such as Daphnia to higher order vertebrates (Weavers & Martin, 2020). Life stage applicability: This key event is not life stage specific (Kalm et al., 2013; Veeraraghan et al., 2011; Hladik & Tapio, 2016).   Sex applicability:  Most studies conducted were on male models, although sex-dependent differences in pro-inflammatory markers have been previously reported (Cekanaviciute et al., 2018; Parihar et al., 2020).   Evidence for perturbation by a prototypic stressor: There is evidence of the increase of pro-inflammatory mediators following perturbation from a variety of stressors including exposure to ionizing radiation. (Abdel-Magied et al., 2019; Cho et al., 2017; Gaber et al., 2003; Ismail et al., 2016; Kim et al. 2002; Lee et al., 2010; Parihar et al., 2018).

Not Specified Unspecific

Not Specified

All life stages

Not Specified Not Specified

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Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42 Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87. Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19. Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. 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(2019), “Mitigating effect of biotin against irradiation-induced cerebral cortical and hippocampal damage in the rat brain tissue”, Environmental Science and Pollution Research, Vol. 26/13, Springer, London, <a href="https://doi.org/10.1007/S11356-019-04806-X" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/S11356-019-04806-X</a>.    Alwine, J. C., D. J. Kemp and G. R. Stark (1977), “Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 74/12, United States National Academy of Sciences, Washington, D.C., <a href="https://doi.org/10.1073/pnas.74.12.5350" rel="noreferrer noopener" target="_blank">https://doi.org/10.1073/pnas.74.12.5350</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> Cekanaviciute, E., S. Rosi and S. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI) AG, Basel,  https://doi.org/10.3390/ijms19113669.   Cho, H. J. et al. (2017), “Role of NADPH Oxidase in Radiation-induced Pro-oxidative and Pro-inflammatory Pathways in Mouse Brain”, International Journal of Radiation Biology, Vol. 93/11, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1377360" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1377360</a>.    Coons, A. H. et al. (1942), “The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody”, The Journal of Immunology, Vol. 45/3, American Association of Immunologists, Minneapolis, pp. 159-169  Engvall, E., and P. Perlmann (1972), “Enzyme-Linked Immunosorbent Assay, Elisa”, The Journal of Immunology, Vol. 109/1, American Association of Immunologists, Minneapolis, pp. 129-135  Fan, L. W. and Y. Pang. (2017), "Dysregulation of neurogenesis by neuroinflammation: Key differences in neurodevelopmental and neurological disorders", Neural Regeneration Research, Vol. 12/3, Wolters Kluwer, Alphen aan den Rijn, <a href="https://doi.org/10.4103/1673-5374.202926" rel="noreferrer noopener" target="_blank">https://doi.org/10.4103/1673-5374.202926</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   Gaber, M. W. et al. 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Halsey (2005), “Measurement of Cytokine Production Using Whole Blood”, in Current Protocols in Immunology, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1002/0471142735.im0718bs66" rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/0471142735.im0718bs66</a>  Veeraraghavan, J. et al. (2011), "Low-dose γ-radiation-induced oxidative stress response in mouse brain and gut: Regulation by NFκB–MnSOD cross-signaling", Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol. 718/1–2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrgentox.2010.10.006.  Veremeyko, T. et al. (2012), “Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation”, Journal of Visualized Experiments: JoVE, Vol. 65, MyJove Corporation, Cambridge, <a href="https://doi.org/10.3791/4097" rel="noreferrer noopener" target="_blank">https://doi.org/10.3791/4097</a> Weavers, H. and P. Martin (2020), “The cell biology of inflammation: From common traits to remarkable immunological adaptations”, Journal of Cell Biology, Vol. 219, Rockefeller University Press, New York, <a href="https://doi.org/10.1083/jcb.202004003" rel="noreferrer noopener" target="_blank">https://doi.org/10.1083/jcb.202004003</a>  AOPWiki 2017-11-28T09:00:54

2023-03-21T15:50:49

Increased, secretion of proinflammatory mediators

Increased proinflammatory mediators

Cellular

Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including <a href="https://bioregistry.io/genecards:IL2">IL-2</a>, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL7">IL-7</a>, <a href="https://bioregistry.io/genecards:IL9">IL-9</a>, <a href="https://bioregistry.io/genecards:IL15">IL-15</a>, <a href="https://bioregistry.io/genecards:IL21">IL-21</a>, <a href="https://bioregistry.io/genecards:IL3">IL-3</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a> and Granulocyte-macrophage colony stimulating factor (<a href="https://bioregistry.io/genecards:CSF2">GM-CSF</a>) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (<a href="https://bioregistry.io/genecards:IL1a">IL-1</a>α , <a href="https://bioregistry.io/genecards:IL1b">IL-1</a>β, <a href="https://bioregistry.io/genecards:IL1ra">IL-1r</a>α, <a href="https://bioregistry.io/genecards:IL18">IL-18</a>, <a href="https://bioregistry.io/genecards:IL36a">IL-36</a>α, <a href="https://bioregistry.io/genecards:IL36b">IL-36</a>β, <a href="https://bioregistry.io/genecards:IL36g">IL-36</a>γ, <a href="https://bioregistry.io/genecards:IL36Ra">IL-36R</a>α, <a href="https://bioregistry.io/genecards:IL37">IL-37</a>), <a href="https://bioregistry.io/genecards:IL6">IL-6 </a>family, Tumor necrosis factor (<a href="https://bioregistry.io/genecards:TNF">TNF</a>) family, <a href="https://bioregistry.io/genecards:IL17">IL-17</a>, and Interferon gamma (<a href="https://bioregistry.io/genecards:IFNg">IFN</a>-γ) (Turner et al., 2014). While <a href="https://bioregistry.io/genecards:IL4">IL-4</a> and <a href="https://bioregistry.io/genecards:IL5">IL-5</a> are considered T helper (Th) cell type 2 response, <a href="https://bioregistry.io/genecards:IFNg">IFN</a>-γ is suggested to be Th1 type response. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, <a href="https://bioregistry.io/genecards:IL1">IL-1</a> family cytokines, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a>, <a href="https://bioregistry.io/genecards:IL6">IL-6</a>, <a href="https://bioregistry.io/genecards:TNFa">TNF</a>-α, IFN-γ are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002). Literature evidence for its perturbation: Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-α, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-κB], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-κB luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte chemoattractant protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).

The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies. Real-time reverse transcription-polymerase chain reaction (qRT-PCR) – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially. Enzyme-linked immunosorbent assays (ELISA) – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample. Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009). Cell models - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used in vitro due to the large literature base, and ease of use, but do not adequately mimic the in vivo condition (Sharma et al., 2016; Sundarakrishnan et al., 2018). Recently, the EpiAlveolar in vitro lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNTs, and researchers noted increases in the pro-inflammatory molecules TNF-α, IL-1β, and the pro-fibrotic TGF-β using ELISA (Barasova et al., 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the in vivo pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments.

Human, mouse, rat Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as a diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.

CL:0000255

CL eukaryotic cell

High Male High Female

High

Adults

High High High

1. Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5.  2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860.  3. Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012.  4. Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548. 5. Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639.  6. Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001. 7. Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003. 8. Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192. 9. Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6. 10. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731. 11. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236. 12. Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P2) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131. 13. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111. 14. Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7.  15. Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7.  16. Sundarakrishnan A, Chen Y, Black LD, Aldridge BB, Kaplan DL. Engineered cell and tissue models of pulmonary fibrosis. Adv Drug Deliv Rev. 2018 Apr;129:78-94. doi: 10.1016/j.addr.2017.12.013. 17. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014.  18. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099. AOPWiki 2018-01-02T13:12:11

2023-05-17T15:18:03

formation of neutrophil extracellular traps (NETs)

NETosis

Cellular

AOPWiki 2021-07-08T07:29:47

2021-07-08T07:29:47

Hyperinflammation

Tissue

Hyperinflammation can be defined as an uncontrolled and self-perpetuating inflammatory process that results in tissue damage. The state of hyperinflammation is also observed in cytokine storm syndrome, cytokine release syndrome, haemophagocytic lymphohistiocytosis, macrophage activation syndrome and in conditions of sepsis; however, it is not a frequent observation. For example, in COVID-19 infection, hyperinflammation plays a critical role in driving the disease severity. Although high viral titre initiates the cascade, the disease severity itself is dependent on the severity of the inflammatory state. Clinically, the hallmarks of hyperinflammation state include excessive serum levels of pro-inflammatory mediator C-reactive protein (<a href="http://bioregistry.io/genecards:CRP">CRP</a>), reduced or absence of lymphocytes (lymphopenia), high levels of ferritin and D-dimer, and increased lactate dehydrogenase. Higher neutrophil to lymphocyte ratio is another clinical marker. Some research studies have also associated high serum levels of<a href="https://bioregistry.io/genecards:IL6"> IL6</a> protein and accumulation of neutrophils to be causal and indicative of hyperinflammation. Other molecular markers associated with hyperinflammation include <a href="http://bioregistry.io/genecards:IL1B">IL1ꞵ</a> and <a href="http://bioregistry.io/genecards:TNFA">TNFɑ</a> and have together with IL6 and a multitude of other cytokines, chemokines and other proinflammatory factors been identified as potential therapeutic targets (<a href="https://doi.org/10.1371/journal.pone.0254374">Desvaux et al. 2021</a>). While the total serum levels of these markers is important, more critically, how fast the levels increase in serum is taken into consideration in judging the severity (<a href="http://doi.org/10.1101/2021.01.11.20248765">Bergamaschi et al. 2021</a>). The number of studies that have reported on the various markers of hyperinflammation is listed in Table-1.   Although the initiation and promotion of inflammation involves several cell types including epithelial cells, alveolar macrophages, type I and II pneumocytes and dendritic cells, the cell types that play role on inducing hyperinflammatory state may include macrophages, dendritic cells and neutrophils. Lack of neutrophil plays an important role in slowing the viral clearance and thus perpetuating the condition. Hyperferritinaemia is associated with high macrophage activation. Weight of evidence KE Hyperinflammation <table border="1" bordercolor="#ccc" cellpadding="5" cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse"> <tbody> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:110px"> References </td> <td colspan="6" style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:396px"> Markers </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:127px"> Comments </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Research </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px"> Clinical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px"> Clinical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> Clinical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px"> Clinical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Research </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> IL6, TNFa </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px"> CRP </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px"> Lymphopenia </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> Ferritin </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px"> Lactate dehydrogenase </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Impaired IFN 1 type response </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Lazear H.M et al., Immunity. 2019;50:907–923.  </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased protein levels, NFkB pathway activation </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Reduced IFN stimulated genes </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Zhang B, Zhou X, Qiu Y, et al. Clinical characteristics of 82 death cases with COVID‐19. medRxiv. 2020. </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px"> Present, also thrombocytopenia </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> IncreasedIncreased D-dimer </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> J Clin.  Invest. 2020;<a href="http://www.jci.org/130/5" style="color:blue; text-decoration:underline">130(5)</a>:2620-2629. <a href="https://doi.org/10.1172/JCI137244" style="color:blue; text-decoration:underline">https://doi.org/10.1172/JCI137244</a> </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px"> Present </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> Increased D-dimer </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Hadjadj et al., Science doi: <a href="http://dx.doi.org/10.1126/science.abc6027" style="color:blue; text-decoration:underline" target="_blank">10.1126/science.abc6027</a> </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increasedprotein </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Impaired </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Del Valle DM et al., Medrxiv : the Preprint Server for Health Sciences. 2020 May.  </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased protein </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Impaired </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Chen G ei al., J Clin Invest. 2020;130(5):2620-2629 </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased IL-6 </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px"> Present </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> Increased ferritin and D-dimer </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Marginal reduction </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Cheng L et al., Journal of Clinical Laboratory Analysis <a href="https://onlinelibrary.wiley.com/toc/10982825/2020/34/10" style="color:blue; text-decoration:underline" title="View Volume 34, Issue 10">Volume34, Issue10</a> October 2020 e23618 <a href="https://doi.org/10.1002/jcla.23618" style="color:blue; text-decoration:underline">https://doi.org/10.1002/jcla.23618</a>   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> IL-6 increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> Increased ferritin levels </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> Review – meta analysis of 52 studies that have data for ferritin levels. Showing severity can be predicted by ferritin levels. Connections with inflammation state. </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Manson JJ et al., <a href="https://www.sciencedirect.com/science/journal/26659913" style="color:blue; text-decoration:underline" title="Go to The Lancet Rheumatology on ScienceDirect">The Lancet Rheumatology</a> <a href="https://www.sciencedirect.com/science/journal/26659913/2/10" style="color:blue; text-decoration:underline" title="Go to table of contents for this volume/issue">Volume 2, Issue 10</a>, October 2020, Pages e594-e602   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px"> Increased Ferritin levels </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> Logitudinal cohort study showing association of hyperinflammation with prognosis. Only CRP and Ferritin levels considered. </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Caricchio R, et al.,  Ann Rheum Dis doi:10.1136/ annrheumdis-2020-218323 </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> Recommended criteria for assessing hyperinflammation </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Mojtabavi, H., et al., Eur Cytokine Netw 31, 44–49 (2020). https://doi.org/10.1684/ecn.2020.0448 </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> Review- meta-analysis of available data. 11 studies included. </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Henry B et al.,  Acta Biomed. 2020;91(3):e2020008. doi:10.23750/abm.v91i3.10217 </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px"> Lymphopenia and neutrophilia </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> Meta-analysis study – 22 studies included. Correlation between lymphopenia and neutrophilia at admission with severity of disease. </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Human </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Jin J-M et al., Front. Public Health, 29 April 2020 | <a href="https://doi.org/10.3389/fpubh.2020.00152" style="color:blue; text-decoration:underline">https://doi.org/10.3389/fpubh.2020.00152</a> </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> Gender differences </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Ex vivo, human lung tissue </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Chu H, et al., Clin Infect Dis. 2020;71(6):1400-1409. </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Increased IL-6 </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Impaired IFN I, II, III signalling </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px">   </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:56px"> Mouse </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:110px"> Channappanavar et al.,  Cell Host Microbe 19 (2) (2016) 181–193, </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:56px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:60px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:94px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:57px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:69px">   </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:59px"> Reduced IFN I response </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:127px"> SARS-COV </td> </tr> </tbody> </table>

Hyperinflammation is observed in all age groups with high rates of infection and mortality observed in aged population. In children, although the rate of infection is low, hyperinflammatory syndrome is observed leading to long term disabilities. However, mortality rate in young children and adults below 40 years of age is less pronounced. Data in other developmental stages is lacking. Prevalence of hyperinflammation is same in men and women; however, studies have found that men develop more severe symptoms than women.

Not Specified Mixed High Moderate

1. Caricchio R, et al.,  Ann Rheum Dis doi:10.1136/ annrheumdis-2020-218323 2. Channappanavar et al.,  Cell Host Microbe 19 (2) (2016) 181–193, 3. Chen G ei al., J Clin Invest. 2020;130(5):2620-2629 4. Cheng L et al.,Journal of Clinical Laboratory Analysis<a href="https://onlinelibrary.wiley.com/toc/10982825/2020/34/10"> Volume34, Issue10</a>, October 2020 e23618, <a href="https://doi.org/10.1002/jcla.23618">https://doi.org/10.1002/jcla.23618</a> 5. Chu H, et al., Clin Infect Dis. 2020;71(6):1400-1409. 6. Del Valle DM et al., Medrxiv : the Preprint Server for Health Sciences. 2020 May. 7. J Clin.  Invest. 2020;<a href="http://www.jci.org/130/5">130(5)</a>:2620-2629. <a href="https://doi.org/10.1172/JCI137244">https://doi.org/10.1172/JCI137244</a> 8. Jin J-M et al., Front. Public Health, 29 April 2020 | <a href="https://doi.org/10.3389/fpubh.2020.00152">https://doi.org/10.3389/fpubh.2020.00152</a> 9. Hadjadj et al., Science doi: <a href="http://dx.doi.org/10.1126/science.abc6027">10.1126/science.abc6027</a> 10. Henry B et al.,  Acta Biomed. 2020;91(3):e2020008. doi:10.23750/abm.v91i3.10217 11. Lazear H.M et al., Immunity. 2019;50:907–923. 12. Manson JJ et al.,<a href="https://www.sciencedirect.com/science/journal/26659913"> The Lancet Rheumatology</a> <a href="https://www.sciencedirect.com/science/journal/26659913/2/10">Volume 2, Issue 10</a>, October 2020, Pages e594-e602 13. Mojtabavi, H., et al., Eur Cytokine Netw 31, 44–49 (2020). https://doi.org/10.1684/ecn.2020.0448 14. Zhang B, Zhou X, Qiu Y, et al. Clinical characteristics of 82 death cases with COVID‐19. medRxiv. 2020. AOPWiki 2021-04-20T02:44:25

2021-12-29T02:29:24

Release, Cytokine

Cellular

Cytokines are small, soluble molecules secreted by cells to enable intercellular communication. Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), as well as on distant cells (endocrine action). Cytokines can act synergistically or antagonistically, and secretion from one cell can trigger upregulation of a further range of cytokines from the same cell or others <a href="#cite_note-1">[1]</a>. Most cells in the body are able to secrete them, and several subfamilies belong to the group of cytokines, such as chemokines, interferons, interleukins, tumor necrosis factors (TNF), transforming growth factors (TGF) and colony-stimulating factors. They are important players in modulating fundamental biological processes, including body growth, adiposity, lactation, hematopoiesis, and also inflammation and immunity<a href="#cite_note-Braunersreuther2012-2">[2]</a>. Damaged cells, such as apoptotic cells, can trigger the upregulation and release of cytokines to induce the inflammatory response. An important receptor responsible for cell death-related cytokine regulation is Fas, a cell surface glycoprotein which belongs to the tumor necrosis factor (TNF) receptor family. The role of Fas in the onset of inflammation by upregulating inflammatory cytokines is increasingly discussed. Fas-activation can trigger the production of MCP-1 and IL-8 and its associated chemotaxis of phagocytes toward apoptotic cells<a href="#cite_note-Cullen2013-3">[3]</a>. TNF-α is an inflammatory mediator that can be secreted by many cell types, including hepatocytes and Kupffer cells. TNF-induced cytokines and chemokines, such as IL-6, IL-8, GMCSF, CXCL1, and RANTES, can trigger immune responses by producing acute phase proteins and recruitment of inflammatory cells such as neutrophils, macrophages, and basophils to the site of inflammation. Moreover, an increased production of monocytes/macrophages from bone marrow is triggered<a href="#cite_note-Cullen2013-3">[3]</a>. On the other hand, inflammation can be suppressed by cytokines and mediators such as IL-10 and TGF-β. In the liver, TGF-β1 is the most abundant isoform and is secreted by immune cells, stellate cells, and epithelial cells. IL-10 inhibits T cell-, monocyte-, and macrophage-mediated functions and has been detected in several liver cells, in¬cluding hepatocytes, stellate cells, and Kupffer cells <a href="#cite_note-Braunersreuther2012-2">[2]</a>.

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? mRNA expression levels of inflammatory cytokines can be determined by using real-time PCR as described in <a href="#cite_note-Cui2011-4">[4]</a>. Equally, In Situ Hybridization of mRNA in liver tissue can be used <a href="#cite_note-Faouzi2001-5">[5]</a>. Plasma levels of pro-inflammatory cytokines, or levels in cell supernatants can be analysed by enzyme linked immunosorbent assay (ELISA) using commercial kits <a href="#cite_note-Ma2009-6">[6]</a><a href="#cite_note-Cullen2013-3">[3]</a>. A more advanced system was described recently by using a multiplex immunoassay platform. In a 96 well plate format the authors describe the analysis of blood, urine and breath samples of human volunteers in a Meso Scale Discovery (MSD) multiplex electrochemiluminescent immunoassay system <a href="#cite_note-Stiegel2015-7">[7]</a>.

<a href="#cite_note-Cui2011-4">[4]</a><a href="#cite_note-Ma2009-6">[6]</a><a href="#cite_note-Faouzi2001-5">[5]</a>: mouse <a href="#cite_note-Cullen2013-3">[3]</a><a href="#cite_note-Stiegel2015-7">[7]</a>: human

CL:0000255

CL eukaryotic cell

High High

<ol class="references"> <li id="cite_note-1"><a href="#cite_ref-1">↑</a> Zhang JM, An J. Cytokines, inflammation, and pain. Int Anesthesiol Clin. 2007 Spring;45(2):27-37 </li> <li id="cite_note-Braunersreuther2012-2">↑ <a href="#cite_ref-Braunersreuther2012_2-0">2.0</a> <a href="#cite_ref-Braunersreuther2012_2-1">2.1</a> Braunersreuther V, Viviani GL, Mach F, Montecucco F. Role of cytokines and chemokines in non-alcoholic fatty liver disease. World J Gastroenterol. 2012 Feb 28;18(8):727-35 </li> <li id="cite_note-Cullen2013-3">↑ <a href="#cite_ref-Cullen2013_3-0">3.0</a> <a href="#cite_ref-Cullen2013_3-1">3.1</a> <a href="#cite_ref-Cullen2013_3-2">3.2</a> <a href="#cite_ref-Cullen2013_3-3">3.3</a> Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ. Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells. Mol Cell. 2013 Mar 28;49(6):1034-48 </li> <li id="cite_note-Cui2011-4">↑ <a href="#cite_ref-Cui2011_4-0">4.0</a> <a href="#cite_ref-Cui2011_4-1">4.1</a> Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229 </li> <li id="cite_note-Faouzi2001-5">↑ <a href="#cite_ref-Faouzi2001_5-0">5.0</a> <a href="#cite_ref-Faouzi2001_5-1">5.1</a> Faouzi S, Burckhardt BE, Hanson JC, Campe CB, Schrum LW, Rippe RA, Maher JJ. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J Biol Chem. 2001 Dec 28;276(52):49077-82 </li> <li id="cite_note-Ma2009-6">↑ <a href="#cite_ref-Ma2009_6-0">6.0</a> <a href="#cite_ref-Ma2009_6-1">6.1</a> Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85 </li> <li id="cite_note-Stiegel2015-7">↑ <a href="#cite_ref-Stiegel2015_7-0">7.0</a> <a href="#cite_ref-Stiegel2015_7-1">7.1</a> Stiegel MA, Pleil JD, Sobus JR, Morgan MK, Madden MC. Analysis of inflammatory cytokines in human blood, breath condensate, and urine using a multiplex immunoassay platform. Biomarkers. 2015 Feb;20(1):35-46 </li> </ol> AOPWiki 2016-11-29T18:41:22

2017-09-16T10:14:42

Increase, Tissue/organ damage

Organ

AOPWiki 2020-11-02T05:40:46

2020-11-02T05:49:53

Increase, the risk of acute respiratory failure

Organ

AOPWiki 2020-03-10T02:05:09

2020-03-10T02:05:09

Heart failure

Organ

AOPWiki 2018-06-19T14:04:03

2018-06-19T14:04:03

Increased, Kidney Failure

Population

AOPWiki 2016-11-29T18:41:26

2019-01-16T08:57:09

Occurrence, Kidney toxicity

Organ

The kidneys are a crucial site of regulation of divalent cation levels in the plasma through filtration, reabsorption, and concentration (cite). On top of their excretion capabilities, the kidneys are also responsible for the production of hormones crucial for hematologic, cardiovascular, and skeletal muscle homeostasis (Bonventre et al., 2010). Nephrons are the functional units of the kidney and each kidney is made up of approximately 1 million nephrons (Bonventre et al., 2010). The nephrons are vital in reabsorption of these cations where 70% of transport has been shown to occur in the proximal tubule (Barbier et al., 2005). The kidneys are thought to be very susceptible to toxicity due to the increased concentration through their filtering structures with the tubular uptake mechanisms, specifically those of the proximal tubule, magnifying intracellular concentrations (Bonventre et al., 2010; Weber et al., 2017). Commonly, biomarkers like serum creatinine (sCr) and blood urea nitrogen (BUN) are utilized to identify kidney toxicity; however, these markers have been identified as nonspecific to the area of the kidney and slow in identification. Bonventre et al. (2010) has explored other biomarkers that may be used to identify segment specific injury. Proximal tubule injury can be identified using: albumin, RPB, NAG, clusterin, osteopontin, a1-microglobulin, and many others. Glomerulus damage can be identified through urinary Cystatin C, b2-microglobulin, a1-microglobulin, albumin, and more (Bonventre et al., 2010). These biomarkers do show some overlap between regions and can indicate damage to various areas of the nephron, though it is important to note the development of these specific techniques and therefore, the ability to develop more tailored and earlier identifying testing procedures. Since there are many essential metals for cellular function, there are also many transporters responsible for facilitating ionic entry into the cell and the designated cellular compartment (cite). Some of these transporters are very specific to a given metal and some are more diverse in the metals they handle, therefore, these transporters can facilitate the transport of toxic metals into the cell, often through mimickery exhibited by those metals (Ballatori, 2002). DMT1 (divalent metal transporter 1) is a strong example of such transporters. The introduction of toxic divalent cations (Cd2+, Pb2+, Pt2+, etc.) is highly problematic in the kidneys due to increased toxicity and occupancy of DMT1 limiting the transport of essential trace elements. DMT1 is an essential transport molecule that is highly expressed in the kidneys, and is responsible for transport of essential trace divalent cations, as well as highly toxic ones; this competition increases strain on the kidneys exposed to toxic heavy metals (Barbier et al., 2005; Ballatori, 2002). DMT1 has been shown to transport Fe, Zn, Mn, Co, Cd, Cu, Ni, and Pb via a proton-coupled, membrane potential dependant mechanism (Ballatori, 2002). Some toxic metals can also enter a cell by forming complexes that mimic endogenous molecules in their structure. Arsenate and vanadate, for example, act as phosphate mimics both for transport and metabolism, assaulting cellular function by the same mechanism as their initial entry; cromate, selenite and molybdate mimic sulfate in a similar way (Ballatori, 2002). Many of the identified transporters fooled by this mimicry have been localized to the brush border membrane of the renal proximal tubule and epithelial cells. Some divalent metals such as Cd, Ba, and Sr have been shown to enter cells through voltage gated calcium channels. Another important example focused on by Ballatori (2002) is the action of inorganic mercury and methyl mercury (MeHg) that were shown to have high affinity for reduced sulfhydryl groups. These groups are seen on the amino acid cysteine, and importantly on glutathione (GSH), a vital enzymatic antioxidant. MeHg mimics methionine to enter the cell, after which it binds to GSH, and interferes with ATP production (Ballatori, 2002). Uranium has been shown to enter the blood rapidly and then either form stable complexes with plasma proteins, due to its high affinity for phosphate, carboxyl and hydroxyl groups, or binds to bicarbonate in the blood (Keith et al., 2013). In the kidneys, uranium can be released from bicarbonate to combine with other small proteins in the kidney tubular walls, disrupting cellular function (Keith et al., 2013). Uranium has been seen to enter the glomerulus, where it is filtered, via endocytosis as UO+2 binding to anionic sites of proximal tubular epithelial brush borders (Shaki et al., 2012). To further understand the mode of action of heavy metals within the kidneys, many studies have been conducted to determine the specific region primarily damaged. It is also important to note that variation of results may be found in some studies as experimental conditions as well as other factors may influence the mode of action of some metals. Zamora et al. (1998) found that kidney function decrease and cytotoxicity increase were correlated with uranium ingestion. However, no glomerular injury was detected, indicating that chronic uranium ingestion in rats (0.004 µg/kg to 9 µg/kg body weight) damages the proximal tubule and not the glomerulus (Zamora et al., 1998). Homma-Takeda et al. (2013) identifies the kidneys as the major site of depleted uranium toxicity. Studying the kidneys of rats of varying ages, exposed to 0.1-2mg/kg uranyl acetate, they found that the younger kidneys did not flush the uranium out as well. Accumulation of uranium and its damages was seen in the S3 segment of the proximal tubules (Homma-Takeda et al., 2013). Shaki et al. (2012), assessed the mechanism of depleted uranium-induced nephrotoxicity that revealed damage to the mitochondria isolated from uranyl acetate treated rat kidney cells. The damage included oxidative stress, mitochondrial swelling, mitochondrial membrane potential collapse, cytochrome C release, impaired ATP production, and damage to the electron transport chain complexes. Utilizing rat renal brush border vesicles, Goldman et al. (2006) found that exposure to uranyl acetate induced decreased rates of glucose transport, in part due to a decreased number of sodium-coupled glucose transporters; this decreased the ability of the kidneys to reabsorb glucose properly. Berradi et al. (2008) assessed the red blood cell (RBC) count of rats drinking water containing 40mg DU/L and found that chronic exposure to DU causes RBC reduction, pointing to nephrotoxicity as the kidneys play a major role in RBC synthesis. Heavy metals consistently aggregate in the kidneys, and more specifically in the S3 segment of the proximal tubules. Evidence also suggests that uranium and other heavy metals induce nephrotoxicity after endocytosis into cells by disrupting the electron transport chain, inducing oxidative stress. The oxidative stress leads to mitochondrial dysfunction followed by, apoptosis at low doses of uranium and necrosis at  high doses of uranium. Finally, this induces renal injury and tissue damage to the proximal tubules, or nephrotoxicity.

<table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <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> Kidney Function Assay Measuring total urinary protein, albumin, transferrin, b2-microglobulin, retinolbinding protein, brush border tubular antigens, N-acetyl-b-Dglucosaminidase activity, serum and urinary creatine (de Burbure et al., 2003) </td> <td>“All analyses of a given parameter were performed under similar experimental conditions in the same laboratories within 6mo of collection. Total urinary protein (Prot-T-U) was determined by the Coomassie blue G250 binding method. Albumin (Alb-U), transferrin (Transf-U), β2-microglobulin (β2m-U), and retinolbinding protein (RBP-U) in urine were quantified by latex immunoassay (Bernard & Lauwerys, 1983). Acceptable limits for precision and accuracy of measurements and external quality controls were the same as those described in the Cadmibel study (Lauwerys et al., 1990). The brush border tubular antigens (BBA-U) were analyzed by a sandwich enzyme-linked immunoassay using monoclonal antibodies (Mutti et al., 1985). The total activity of N-acetyl-β-Dglucosaminidase (NAG-T-U) in urine was determined colorimetrically using a kit (PPR Diagnostics Ltd.) as described elsewhere (Price et al., 1996). Only total NAG (NAG-T) was used for the purpose of this study. Serum and urinary creatinine (Creat-U) were measured by the methods of Heinegard and Tiderström (1973), and Jaffé, respectively (Henry, 1965).” (de Burbure et al., 2003)</td> <td>“The soil contamination in the area varied from 100 to 1700ppm lead (with values higher than 1000ppm in the immediate vicinity of the factories), 0.7 to 233ppm cadmium, and 101 to 22,257ppm zinc, with the highest concentrations being recorded within 500 m of the 2 factories”</td> <td> </td> </tr> <tr> <td> NAG Assay Measuring N-acetyl-b-D-Glucosaminidase urinary content (Lim et al., 2016)</td> <td>“Urinary NAG activity was measured by using NAG Quantitative Kit (Shionogi, Osaka, Japan). After storing a synthetic substrate solution (1 mL) at 37°C for five minutes, the solution was mixed with the supernatant of the urine samples (50 mL) received after centrifugation. After storing it at 37°C for 15 min, stopping solution (2 mL) was added to and mixed with it. By using a spectrophotometer, its fluorescence intensities were measured with a wavelength of 580 nm (<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780232/#b13-tr-32-057" style="color:#0563c1; text-decoration:underline">13</a>,<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780232/#b14-tr-32-057" style="color:#0563c1; text-decoration:underline">14</a>). Urinary β2-MG was measured by using Enzygnost β2-MG Micro Kit (Behring Institute, Mannheim, Germany). Its method used the principle of solid phase enzyme-linked immunosorbent assay (ELISA). Monoclonal anti-β2-MG antibody and anti-2-MG-horseradish peroxidase conjugate solution were used. After that, color intensities were measured with a wavelength of 450 nm by using a spectrophotometer (<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780232/#b13-tr-32-057" style="color:#0563c1; text-decoration:underline">13</a>,<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780232/#b14-tr-32-057" style="color:#0563c1; text-decoration:underline">14</a>).” (Lim et al., 2016)</td> <td> Pb: 0.0221ppm (converted from blood Pb µg/dL) Cd: 1.08ppm (converted from Urinary Cd μg/g creatinine)</td> <td>Fast, easy, accurate</td> </tr> <tr> <td> Kidney Dysfunction Assay Measuring BUN and creatinine serum blood levels (Shaki et al., 2012)</td> <td>“For studies in vivo rats were fasted overnight, then animals were divided into two groups, with six rats in each group. The control group (vehicle) received a single intraperitoneal (i.p.) injection of saline solution (1 ml per 100 g body weight). Uranyl acetate was dissolved in normal saline. Rats were treated with single intraperitoneal (i.p.) injections of UA in doses 0.5, 1 and 2 mg/kg body weight. These dosages was selected based on previous studies [28], which is sufficient to induce oxidative stress in kidney without causing death and none died within the duration of experiments. Blood urea nitrogen (BUN) and creatinine, marker of kidney dysfunction, were determined by commercial reagents (obtained from Parsazmoon Co., Iran). The rats were killed by decapitation 24 h after injection. The kidney were immediately removed and placed in ice-cold mitochondria isolation medium (0.225 M D-mannitol, 75 mM sucrose, and 0.2 mM EDTA, pH=7.4)” (Shaki et al., 2012)</td> <td>Control, 0.5, 1, 2 mg/kg Uranyl Acetate (UA) </td> <td>Fast, easy, medium accuracy </td> </tr> </tbody> </table>

Higher order animals (mammals) with functional and complete kidneys

UBERON:0002113

UBERON kidney

Not Specified Not Specified

Al Dera, H. S. (2016). Protective effect of resveratrol against aluminum chloride induced nephrotoxicity in rats. Saudi Med J, 37(4), 369-378. doi:10.15537/smj.2016.4.13611 Andjelkovic, M., Djordjevic, A. B., Antonijevic, E., Antonijevic, B., Stanic, M., Kotur-Stevuljevic, J., . . . Bulat, Z. (2019). Toxic effect of acute cadmium and lead exposure in rat blood, liver, and kidney. International Journal of Environmental Research and Public Health, 16, 247. doi:10.3390/ijerph16020274 Arzuaga , X., Rieth, S. H., Bathija, A. & Cooper, G. S. (2010) Renal Effects of Exposure to Natural and Depleted Uranium: A Review of the Epidemiologic and Experimental Data, Journal of Toxicology and Environmental Health, Part B, 13:7-8, 527-545, DOI:10.1080/10937404.2010.509015 Ballatori, N. (2002). Transport of toxic metals by molecular mimicry. Environmental Health Perspectives, 110, 689-694. doi:10.1289/ehp.02110s5689 Barnes, P., Yeboah, J. K., Gbedema, W., Saahene, R. O., & Amoani, B. (2020). Ameliorative effect of vernonia amygdalina plant extract on heavy metal-induced LIver and kidney dysfunction in rats. Advances in Pharmacological and Pharmaceutical Sciences, 2020, 1-7. doi:10.1155/2020/2976905 Barbier, O., Jcquillet, G., Tauc, M., Cougnon, M., & Poujeol, P. (2005). Effect of heavy metals on, and handling by, the  kidney. Nephron Physiology, 99, 105-110. doi:10.1159/000083981 Bonventre, J. V., Vaidya, V. S., Schmouder, R., Feig, P., & Dieterle, F. (2010). Next-generation biomarkers for detecting kidney toxicity. Nature biotechnology, 28(5), 436–440. <a href="https://doi.org/10.1038/nbt0510-436" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/nbt0510-436</a> Brzoska, M. M., Kaminski, M., Supernak-Bobko, D., Zwierz, K., & Moniuszko-Jakoniuk, J. (2003). Changes in the strucutre and function of the kidney of rats chronically exposed to cadmium. I. biochemical and histopathological studies. Arch.Toxicol., 77, 344-352. doi:10.1007/s00204-003-0451-1 Buelna-Chontal, M., Franco, M., Hernandez-Esquivel, L., Pavon, N., Rodriguez-Zalvala, J. S., Correa, F., . . . Chavez, E. (2017). CDP-choline circumvents mercury-induced mitochondrial damage and renal dysfunction. Cell Biology International, 41, 1356-1366. doi:10.1002/cbin.10871 Chtourou, Y., Garoui, E. m., Boudawara, T., & Zeghal, N. (2014). Protective role of silymarin against manganese-induced nephrotoxicity and oxidative stress in rat. Environ Toxicol, 29, 1147-1154. doi:10.1002/tox.21845 Durante, P., Romero, F., Perez, M., Chavez, M., & Parra, G. (2010). Effect of uric acid on nephrotoxicity induced by mercuric chloride in rats. Toxicology and Industrial Health, 26(3), 163-174. doi:10.1177/0748233710362377 García-Niño, W. R., Tapia, E., Zazueta, C., Zatarain-Barrón, Z. L., Hernández-Pando, R., Vega-García, C. C., & Pedraza-Chaverrí, J. (2013). Curcumin pretreatment prevents potassium dichromate-induced hepatotoxicity, oxidative stress, decreased respiratory complex I activity, and membrane permeability transition pore opening. Evidence-Based Complementary and Alternative Medicine, (424692), 1-19. doi:10.1155/2013/424692 Goldman, M., Yaari, A., Doshnitzki, Z., Cohen-Luria, R., & Moran, A. (2006). Nephrotoxicity of uranyl acetate: Effect on rat kidney brush border membrane vesicles. Archives of Toxicology, 80(7), 387-393. doi:10.1007/s00204-006-0064-6 Homma-Takeda S, Kokubo T, Terada Y, Suzuki K, Ueno S, Hayao T, Inoue T, Kitahara K, Blyth BJ, Nishimura M, Shimada Y. Uranium dynamics and developmental sensitivity in rat kidney. J Appl Toxicol. 2013 Jul;33(7):685-94. doi: 10.1002/jat.2870. Epub 2013 Apr 26. PMID: 23619997. Keith, S., Faroon, O., N., R., Scinicariello, F., Wilbur, S., Ingerman, L., . . . Diamond, G. (2013). Toxicological profile for uranium. U.S. Department of Health and Human Services. Agency for Toxic Substances and Disease Registry. Kharroubi, W., Dhibi, M., Mekni, M., Haouas, Z., Chreif, I., Neffati, F., . . . Sakly, R. (2014). Sodium arsenate induce changes in fatty acids profiles and oxidative damage in kidney of rats. Environ Sci Pollut Res, 21, 12040-12049. doi:10.1007/s11356-014-3142-y Lunyera, J., & Smith, S. R. (2017). Heavy metal nephropathy: Considerations for exposure analysis. Kidney International, 92, 548-550. doi:http://dx.doi.org/10.1016/j.kint.2017.04.043 Sabath, E., & Robles-Osorio, M. L. (2012). Renal health and the environment: Heavy metal nephrotoxicity. Revista Nefrologia, doi:10.3265/Nefrologia.pre2012.Jan.10928 Santos, N. A. G., Catão, C. S., Martins, N. M., Curti, C., Bianchi, M. L. P., & Santos, A. C. (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Archives of Toxicology, 81(7), 495-504. doi:10.1007/s00204-006-0173-2 Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015 Soussi, A., Gargouri, M., & El Feki, A. (2018). Effects of co-exposure to lead and zinc on redox status, kidney variables and histopathology in adult albino rats. Toxicology and Industrial Health, 34(7), 469-480. doi:10.1177/0748233718770293 Spreckelmeyer, S., Estrada-Ortiz, N., Prins, G. G. H., van der Zee, M., Gammelgaard, B., Sturup, S., . . . Casini, A. (2017). On the toxicity and transportation mechanisms of cisplatin in kidney tissues in comparison to a gold-based cytotoxic agent. Metallomics, 9, 1786. doi:10.1039/c7mt00271h Turk, E., Kandemir, F. M., Yildirim, S., Caglayan, C., Kucukler, S., & Kuzu, M. (2019). Protective effect of hesperidin on sodium arsenite-induced nephrotoxicity and hepatotoxicity in rats. Biological Trace Element Research, 189, 95-108. doi:10.1007/s12011-018-1443-6 Weber, E. J., Himmelfarb, J., & Kelly, E. J. (2017). Concise review: Current emerging biomarkers of nephrotoxicity. Curr Opin Toxicol., 4, 16-21. doi:10.1016/j.cotox.2017.03.002 Yeh, Y., Lee, Y., Hsieh, Y., & Hwang, D. (2011). Dietary taurine reduces zinc-induced toxicity in male wistar rats. Journal of Food Science, 76(4), 90-98. doi:10.1111/j.1750-3841.2011.02110.x Zamora, L. M., Tracy, B. L., Zielinski, J. M., Meyerhof, D. P., & Moss, M. A. (1998). Chronic ingestion of uranium in drinking water: A study of kidney bioeffects in humans. Toxicological Sciences, 43(1), 68-77. doi:10.1006/toxs.1998.242 AOPWiki 2016-11-29T18:41:27

2022-03-04T10:58:19

Increase activation, Nuclear factor kappa B (NF-kB)

Cellular

The NF-kB pathway consists of a series of events where the transcription factors of the NF-kB family play a key role. The proinflammatory cytokine (IL-1beta) can be activated by NF-kB , including Reactive Oxygen Species produced by  NADPH oxidase (NOX). Upon pathway activation, the IKK complex will be phosphorylated, which in turn phosphorylates IkBa. There, this transcription factor can express pro-inflammatory and pro-fibrotic genes. This can be achieved by ROS, IKK enhancer or nuclear translocation enhancer.

NF-kB transcriptional activity: Beta lactamase reporter gene assay (Miller et al. 2010). NF-kB transcription: Lentiviral NF-kB GFP reporter with flow cytometry (Moujalled et al. 2012) NF-κB translocation: RelA-GFP reporter assay (Frederiksson 2012) (Huppelschoten 2017) IκBa phosphorylation: Western blotting (Miller et al. 2010) NF-κB p65 (Total/Phospho) ELISA ELISA for IL-6, IL-8, and Cox

The ROS directly influences NF-κB signalling, resulting in differential production of cytokines and chemokines (McKay and Cidlowski, 1999; Pernis, 2007). NF-κB regulates pro-inflammatory responses that are transcriptionally mediated by NF‑κB.

UBERON:0000479

UBERON tissue

CL:0000066

CL epithelial cell

Not Specified Mixed

Moderate

Not Otherwise Specified

High

AOPWiki 2016-11-29T18:41:30

2021-03-30T13:17:17

Inflammation, Liver

Organ

Approximately 29 million people in the European Union suffer from a chronic liver condition <a href="#cite_note-Blachier2013-1">[1]</a>. Inflammation is a crucial link that is related to many of these conditions, with the potential for the development of cirrhosis or primary liver cancer which represent the end-stage of liver pathology and are often associated with mortality: chronic hepatitis (A-E), non-alcoholic steatohepatitis (NASH) which is the progressive form of non-alcoholic fatty liver disease (NAFLD), primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) <a href="#cite_note-Blachier2013-1">[1]</a>. Drug-induced liver injury (DILI) still is a major problem in drug development as its early detection is problematic, and acute liver inflammation is the most common symptom. DILI is the main cause for withdrawal of drugs from the pharmaceutical market <a href="#cite_note-2">[2]</a>. Liver inflammation is marked by an increased influx of neutrophils, following the secretion of signaling factors such as CXC chemokines and macrophage inflammatory protein 2 (MIP-2) from damaged cells <a href="#cite_note-3">[3]</a>. Kupffer cells (KCs), the resident macrophages of the liver and accounting for about 15-20% of total cell numbers in a healthy liver. They are the gatekeepers in the liver, as they monitor the blood that enters this organ <a href="#cite_note-Kermanizadeh2012-4">[4]</a><a href="#cite_note-Arrese2016-5">[5]</a>. Activation of KCs by activation of toll like receptors, for example, leads to the recruitment of further inflammatory cells as well as amplified KC activation. This, in turn, activates Hepatic stellate cells (HSCs) <a href="#cite_note-Arrese2016-5">[5]</a> which can link liver inflammation to further severe outcomes such as development of fibrosis A list of drugs generally known to induce DILI can be found here <a href="#cite_note-6">[6]</a>.

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? Liver inflammation is usually confirmed by analysis of histological features, marked by influx of inflammatory cells (mainly neutrophils) which can be stained by using Haematoxylin and eosin <a href="#cite_note-Huebsch2006-7">[7]</a>. In mice, neutrophil influx can be analysed using a mouse MPO ELISA kit for lysed tissue <a href="#cite_note-Kermanizadeh2012-4">[4]</a>. mRNA expression levels of inflammatory cytokines in tissue samples can be determined by using real-time PCR as described in <a href="#cite_note-Cui2011-8">[8]</a>. Plasma levels of pro-inflammatory cytokines can be analysed by enzyme linked immunosorbent assay) ELISA using commercial kits <a href="#cite_note-Ma2009-9">[9]</a>.

<a href="#cite_note-Huebsch2006-7">[7]</a>: human (representative for general application in patients, as liver inflammation is commonly found in patients with DILI) <a href="#cite_note-Cui2011-8">[8]</a><a href="#cite_note-Kermanizadeh2012-4">[4]</a><a href="#cite_note-Ma2009-9">[9]</a>: mouse (nanomaterial-induced) <a href="#cite_note-10">[10]</a>: rat (nanomaterial-induced)

UBERON:0002107

UBERON liver

High High Moderate

<ol class="references"> <li id="cite_note-Blachier2013-1">↑ <a href="#cite_ref-Blachier2013_1-0">1.0</a> <a href="#cite_ref-Blachier2013_1-1">1.1</a> Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol. 2013 Mar;58(3):593-608 </li> <li id="cite_note-2"><a href="#cite_ref-2">↑</a> Larrey D. Epidemiology and individual susceptibility to adverse drug reactions affecting the liver. Semin Liver Dis. 2002;22(2):145-55 </li> <li id="cite_note-3"><a href="#cite_ref-3">↑</a> Jaeschke H. Inflammation in response to hepatocellular apoptosis. Hepatology. 2002 Apr;35(4):964-6 </li> <li id="cite_note-Kermanizadeh2012-4">↑ <a href="#cite_ref-Kermanizadeh2012_4-0">4.0</a> <a href="#cite_ref-Kermanizadeh2012_4-1">4.1</a> <a href="#cite_ref-Kermanizadeh2012_4-2">4.2</a> Kermanizadeh A, Brown DM, Hutchison GR, Stone V. Engineered Nanomaterial Impact in the Liver following Exposure via an Intravenous Route–The Role of Polymorphonuclear Leukocytes and Gene Expression in the Organ. Journal of Nanomed & Nanotechnol 2012;04(01):1–7 </li> <li id="cite_note-Arrese2016-5">↑ <a href="#cite_ref-Arrese2016_5-0">5.0</a> <a href="#cite_ref-Arrese2016_5-1">5.1</a> Arrese M, Cabrera D, Kalergis AM, Feldstein AE. Innate Immunity and Inflammation in NAFLD/NASH. Dig Dis Sci. 2016 May;61(5):1294-303 </li> <li id="cite_note-6"><a href="#cite_ref-6">↑</a> Ortega-Alonso A, Stephens C, Lucena MI, Andrade RJ. Case Characterization, Clinical Features and Risk Factors in Drug-Induced Liver Injury. Int J Mol Sci. 2016 May 12;17(5) </li> <li id="cite_note-Huebsch2006-7">↑ <a href="#cite_ref-Huebsch2006_7-0">7.0</a> <a href="#cite_ref-Huebsch2006_7-1">7.1</a> Huebscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathol. 2006;49:450–465 </li> <li id="cite_note-Cui2011-8">↑ <a href="#cite_ref-Cui2011_8-0">8.0</a> <a href="#cite_ref-Cui2011_8-1">8.1</a> Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229 </li> <li id="cite_note-Ma2009-9">↑ <a href="#cite_ref-Ma2009_9-0">9.0</a> <a href="#cite_ref-Ma2009_9-1">9.1</a> Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85 </li> <li id="cite_note-10"><a href="#cite_ref-10">↑</a> Alarifi S, Ali ., Al-Doaiss AA, Ali BA, Ahmed M, Al-Khedhairy AA. Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats. Intern J Nanomed. 2013;8:3937–3943 </li> </ol> AOPWiki 2016-11-29T18:41:27

2017-09-16T10:16:41

increased platelet surface TLR9 expression

increased platelet tlr9 expression

Cellular

AOPWiki 2021-07-08T09:20:46

2021-07-08T09:22:20

Increased, recruitment of inflammatory cells

Recruitment of inflammatory cells

Tissue

Pro-inflammatory cells originate in bone marrow and are recruited to the site of infection or injury via circulation following specific pro-inflammatory mediator (cytokine and chemokine) signalling. Pro-inflammatory cells are recruited to lungs to clear the invading pathogen or the toxic substance. Monocytes (dendritic cells, macrophages, and neutrophils) are subsets of circulating white blood cells that are involved in the immune responses to pathogen or toxicant stimuli (Kolaczkowska and Kubes, 2013; Kopf et al., 2015). They are derived from the bone marrow. They can differentiate into different macrophage types and dendritic cells. They can be categorised based on their size, the type of cell surface receptors and their ability to differentiate following external or internal stimulus such as increased expression of cytokines. Monocytes participate in tissue healing, clearance of toxic substance or pathogens, and in the initiation of adaptive immunity. Recruited monocytes can also influence pathogenesis (Ingersoll et al., 2011). Sensing or recognition of pathogens and harmful substances results in the recruitment of monocytes to lungs (Shi and Pamer, 2011). Activated immune cells secrete a variety of pro-inflammatory mediators, the purpose of which is to propagate the immune signalling and response, which when not controlled, leads to chronic inflammation, cell death and tissue injury. Thus, Event 1496 and Event 1497 act in a positive feedback loop mechanism and propagate the proinflammatory environment. Literature evidence for its perturbation: Macrophages accumulate in bronchoalveolar fluid (BALF) post-exposure to bleomycin (Phan et al., 1980; Smith et al., 1995). Nanomaterial (NM)-induced inflammation is predominantly neutrophilic (Poulsen et al., 2015; Rahman L et al., 2017a; Rahman et al., 2017b; Shvedova et al., 2005). An increased number of neutrophils (Reynolds et al., 1977) is observed in the BALF of patients with idiopathic pulmonary fibrosis. Eosinophils are a type of white blood cells and a type of granulocytes (contain granules and enzymes) that are recruited following exposure to allergens, during allergic reactions such as asthma or during fibrosis (Reynolds et al., 1977). Multi-walled carbon nanotubes (MWCNTs) induce increased eosinophil count in lungs (Købler C et al., 2015). MWCNTs act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer et al., 2013). It is important to note that the stressor-induced Event 1495, Event 1496, and Event 1497 are part of the functional changes that we collectively consider as inflammation, and together, they mark the initiation of acute inflammatory phase. Event 1495 and Event 1496 occur at the cellular level. Event 1497 occurs at the tissue level.

In vivo, recruitment of pro-inflammatory cells is measured using BALF cellularity assay. The fluid lining the lung epithelium is lavaged (BALF) and its composition is assessed as marker of lung immune response to the toxic substances or pathogens. BALF is assessed quantitatively for types of infiltrating cells, levels and types of cytokines and chemokines. Thus, BALF assessment can aid in developing dose-response of a substance, to rank a substances’ potency and to set up no effect level of exposure for the regulatory decision making. For NMs, in vivo BALF assessment is recommended as a mandatory test (discussed in ENV/JM/MONO(2012)40 and also in OECD inhalation test guideline for NMs). Temporal changes in the BALF composition can be prognostic of initiation and progression of lung immune disease (Cho et al., 2010). In vitro, it is difficult to assess the recruitment of pro-inflammatory cells. Thus, a suit of pro-inflammatory mediators specific to cell types are assessed using the same techniques mentioned above (real-time reverse transcription-polymerase chain reaction [qRT-PCR], enzyme-linked immunosorbent assays [ELISA], immunohistochemistry) in cell culture models, as indicative of recruitment of cells into the lungs. Alternatively, the use of precision cut lung slices can allow for limited assessment of recruitment of tissue resident inflammatory cells, based on the repertoire of cells remaining in the specific slice following harvesting. This method was used to show that there is a histological increase in inflammatory foci following treatment with bleomycin and MWCNTs (Rahman et al., 2020). Finally, more complicated microfluidic lung-on-a-chip devices can be used to assess the migration of select immune cells and fibroblasts toward a simulated epithelium following treatment with a pro-fibrotic compound (He et al., 2017). However, this method is limited to two cell types, and it lacks the reservoirs of immune cells present in the body in vivo.

Human, mouse, rat

High Mixed

High

All life stages

High High High

1. Beamer CA, Girtsman TA, Seaver BP, Finsaas KJ, Migliaccio CT, Perry VK, Rottman JB, Smith DE, Holian A. IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung. Nanotoxicology. 2013 Sep;7(6):1070-81. doi: 10.3109/17435390.2012.702230. 2. Cho WS, Duffin R, Poland CA, Howie SE, MacNee W, Bradley M, Megson IL, Donaldson K. Metal oxide nanoparticles induce unique inflammatory footprints in the lung: important implications for nanoparticle testing. Environ Health Perspect. 2010 Dec;118(12):1699-706. doi: 10.1289/ehp.1002201.  3. He J, Chen W, Deng S, Xie L, Feng J, Geng J, et al. Modeling alveolar injury using microfluidic co-cultures for monitoring bleomycin-induced epithelial/fibroblastic cross-talk disorder. RSC Advances. 2017 7(68):42738-49. doi: 10.1039/C7RA06752F. 4. Ingersoll MA, Platt AM, Potteaux S, Randolph GJ. Monocyte trafficking in acute and chronic inflammation. Trends Immunol. 2011 Oct;32(10):470-7. doi: 10.1016/j.it.2011.05.001. 5. Købler C, Poulsen SS, Saber AT, Jacobsen NR, Wallin H, Yauk CL, Halappanavar S, Vogel U, Qvortrup K, Mølhave K. Time-dependent subcellular distribution and effects of carbon nanotubes in lungs of mice. PLoS One. 2015 Jan 23;10(1):e0116481. doi: 10.1371/journal.pone.0116481.  6. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013 Mar;13(3):159-75. doi: 10.1038/nri3399.  7. Kopf M, Schneider C, Nobs SP. The development and function of lung-resident macrophages and dendritic cells. Nat Immunol. 2015 Jan;16(1):36-44. doi: 10.1038/ni.3052. 8. Phan SH, Thrall RS, Ward PA. Bleomycin-induced pulmonary fibrosis in rats: biochemical demonstration of increased rate of collagen synthesis. Am Rev Respir Dis. 1980 Mar;121(3):501-6. doi: 10.1164/arrd.1980.121.3.501.  9. Poulsen SS, Saber AT, Williams A, Andersen O, Købler C, Atluri R, Pozzebon ME, Mucelli SP, Simion M, Rickerby D, Mortensen A, Jackson P, Kyjovska ZO, Mølhave K, Jacobsen NR, Jensen KA, Yauk CL, Wallin H, Halappanavar S, Vogel U. MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol. 2015 Apr 1;284(1):16-32. doi: 10.1016/j.taap.2014.12.011.  10. Rahman L, Wu D, Johnston M, William A, Halappanavar S. Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses. Mutagenesis. 2017a Jan;32(1):59-76. doi: 10.1093/mutage/gew048.  11. Rahman L, Jacobsen NR, Aziz SA, Wu D, Williams A, Yauk CL, White P, Wallin H, Vogel U, Halappanavar S. Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis. Mutat Res Genet Toxicol Environ Mutagen. 2017b Nov;823:28-44. doi: 10.1016/j.mrgentox.2017.08.005.  12. 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.  13. Reynolds HY, Fulmer JD, Kazmierowski JA, Roberts WC, Frank MM, Crystal RG. Analysis of cellular and protein content of broncho-alveolar lavage fluid from patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis. J Clin Invest. 1977 Jan;59(1):165-75. doi: 10.1172/JCI108615. 14. Shi C, Pamer EG. Monocyte recruitment during infection and inflammation. Nat Rev Immunol. 2011 Oct 10;11(11):762-74. doi: 10.1038/nri3070.  15. Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, Tyurina YY, Gorelik O, Arepalli S, Schwegler-Berry D, Hubbs AF, Antonini J, Evans DE, Ku BK, Ramsey D, Maynard A, Kagan VE, Castranova V, Baron P. Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L698-708. doi: 10.1152/ajplung.00084.2005.   16. Smith RE, Strieter RM, Zhang K, Phan SH, Standiford TJ, Lukacs NW, Kunkel SL. A role for C-C chemokines in fibrotic lung disease. J Leukoc Biol. 1995 May;57(5):782-7. doi: 10.1002/jlb.57.5.782.  AOPWiki 2018-01-03T09:31:07

2023-05-12T17:03:00

N/A, Mitochondrial dysfunction 1

Cellular

Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress. Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006). Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation. A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010). Summing up: Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.

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

Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010).

CL:0000255

CL eukaryotic cell

Not Specified Unspecific

Not Specified

All life stages

High High High

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Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies. J Pharmacol Toxicol Methods. 2014 Jul-Aug;70(1):40-7. Fiskum G. Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma. 2000 Oct;17(10):843-55. Friberg H, Wieloch T. (2002). Mitochondrial permeability transition in acute neurodegeneration. Biochimie 84:241–250. Fujikawa DG, The Role of Excitotoxic Programmed Necrosis in Acute Brain Injury. Computational and Structural Biotechnology Journal, 2015, 13: 212–221. Graier WF, Frieden M, Malli R. (2007). Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375–396. Green DR. (1998). Apoptotic pathways: the roads to ruin. Cell 94:695-698. Grivennikova VG, Vinogradov AD. Generation of superoxide by the mitochondrial Complex I. Biochim Biophys Acta. 2006, 1757(5-6):553-61. Hafner RP, Brown GC, Brand MD: Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top-down’ approach of metabolic control theory. Eur J Biochem188 :313 –319,1990. Hao, Y., Huang, J., Liu, C., Li, H., Liu, J., Zeng, Y., . . . Li, R. (2016). Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity. Scientific Reports, doi:10.1038/srep38942 Hao, Y., Ren, J., Liu, C., Li, H., Liu, J., Yang, Z., . . . Su, Y. (2014). Zinc protects human kidney cells from depleted uranium induced apoptosis. Basic & Clinical Pharmacology & Toxicology, 114, 271-280. doi:10.1111/bcpt.12167 Hinkle PC (1995) Measurement of ADP/O ratios. In Bioenergetics: A Practical Approach. Brown GC, Cooper CE, Eds. Oxford, U.K., IRL Press, p.5 –6. Huerta-García, E., Perez-Arizti, J. A., Marquez-Ramirez, S. G., Delgado-Buenrostro, N. L., Chirino, Y. I., Iglesias, G. G., & Lopez-Marure, R. (2014). Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells. Free Radical Biology and Medicine, 73, 84-94. doi:10.1016/j.freeradbiomed.2014.04.026 Hüser, J., Rechenmacher, C. E., & Blatter, L. A. (1998). Imaging the permeability pore transition in single mitochondria. Biophysical Journal, 74(4), 2129-2137. doi:10.1016/S0006-3495(98)77920-2 Hynes, J.. Marroquin, L.D Ogurtsov, V.I. Christiansen, K.N. Stevens, G.J. Papkovsky, D.B. Will, Y. (2006)) Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes, Toxicol. Sci. 92 186–200. James, P.E. Jackson, S.K.. Grinberg, O.Y Swartz, H.M. (1995) The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study, Free Radic. Biol. Med. 18 (1995) 641–647. Kang J, Pervaiz S. (2012). Mitochondria: Redox Metabolism and Dysfunction. Biochem Res Int 2012:896751. Kann O, Kovács R. (2007). Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641-576. Karlsson, H. L., Gustafsson, J., Cronholm, P., & Möller, L. (2009). Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters, 188(2), 112-118. doi:<a href="https://doi.org/10.1016/j.toxlet.2009.03.014" target="_blank">10.1016/j.toxlet.2009.03.014</a> Knott Andrew B., Guy Perkins, Robert Schwarzenbacher & Ella Bossy-Wetzel. Mitochondrial fragmentation in neurodegeneration. Nature Reviews Neuroscience, 2008, 229: 505-518. Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649. Llaudet E, Hatz S, Droniou M, Dale N. Microelectrode biosensor for real-time measurement of ATP in biological tissue. Anal Chem. 2005, 77(10):3267-73. Lee HC, Wei YH. (2012). Mitochondria and aging. Adv Exp Med Biol 942:311-327. Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem.2003;278:8516–8525. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006. 443:787-795. Martin LJ. (2011). Mitochondrial pathobiology in ALS. J Bioenerg Biomembr 43:569 – 579. Martinez-Cruz, Oliviert Sanchez-Paz, Arturo Garcia-Carreño, Fernando Jimenez-Gutierrez, Laura Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, <a class="internal mw-magiclink-isbn" href="/wiki/index.php/Special:BookSources/9789535100904">ISBN 978-953-51-0090-4</a>, Publisher InTech, 2012, 181-218. McBride HM, Neuspiel M, Wasiak S. (2006). Mitochondria: more than just a powerhouse. Curr Biol 16:R551–560. McCord, J.M. and I. Fidovich (1968) The Reduction of Cytochrome C by Milk Xanthine Oxidase. J. Biol. Chem. 243:5733-5760. Mei Y, Thompson MD, Cohen RA, Tong X. (2013) Endoplasmic Reticulum Stress and Related Pathological Processes. J Pharmacol Biomed Anal.. 1:100-107. Miccadei, S., & Floridi, A. (1993). Sites of inhibition of mitochondrial electron transport by cadmium. Elsevier Scientific Publishers Ireland Ltd., 89, 159-167.Xu, X. M., & Møller, S. G. (2010). ROS removal by DJ-1: Arabidopsis as a new model to understand Parkinson's Disease. Plant signaling & behavior, 5(8), 1034–1036. doi:10.4161/psb.5.8.12298 Modjtahedi N, Giordanetto F, Madeo F, Kroemer G. Apoptosis-inducing factor: vital and lethal. Trends Cell Biol. 2006 May;16(5):264-72. Nunnari J, Suomalainen A. (2012). Mitochondria: in sickness and in health. Cell 148:1145–1159. Hajnóczky G, Csordás G, Das S, Garcia-Perez C, Saotome M, Sinha Roy S, Yi M. (2006). Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40:553-560. Oliviert Martinez-Cruz, Arturo Sanchez-Paz, Fernando Garcia-Carreño, Laura Jimenez-Gutierrez, Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, <a href="/wiki/index.php/Special:BookSources/9789535100904">ISBN 978-953-51-0090-4</a>, Publisher InTech, 2012, 181-218. Owen, J. B., & Butterfield, D. A. (2010). Measurement of oxidized/reduced glutathione ratio. Methods in Molecular Biology (Clifton, N.J.), 648, 269-277. doi:10.1007/978-1-60761-756-3_18 [doi] Owens R.G. and King F.D. The measurement of respiratory lectron-transport system activity in marine zooplankton. Mar. Biol. 1975, 30:27-36. Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466 Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F: Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999, 76:725-734. Pozzan, T., & Rudolf, R. (2009). Measurements of mitochondrial calcium in vivo. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1787(11), 1317-1323. doi:<a href="https://doi.org/10.1016/j.bbabio.2008.11.012" target="_blank">https://doi.org/10.1016/j.bbabio.2008.11.012</a> Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI. Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379. Radkowsky, A.E. and E.M. Kosower (1986) Bimanes 17. (Haloalkyl)-1,5-diazabicyclo[3.3.O]octadienediones (halo-9,10- dioxabimanes): reactivity toward the tripeptide thiol, glutathione, J. Am. Chem. Soc 108:4527-4531. Ruch, W., P.H. Cooper, and M. Baggiollini (1983) Assay of H2O2 production by macrophages and neutrophils with Homovanillic acid and horseradish peroxidase. J. Immunol Methods 63:347-357. Sanders LH, McCoy J, Hu X, Mastroberardino PG, Dickinson BC, Chang CJ, Chu CT, Van Houten B, Greenamyre JT. (2014a). Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease. Neurobiol Dis. 70:214-23. Sanders LH, Howlett EH2, McCoy J, Greenamyre JT. (2014b) Mitochondrial DNA damage as a peripheral biomarker for mitochondrial toxin exposure in rats. Toxicol Sci. Dec;142(2):395-402. Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015 Shaki, F., Hosseini, M., Ghazi-Khansari, M., & Pourahmad, J. (2013). Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria. Metallomics, 5(6), 736-744. doi:10.1039/c3mt00019b Single B, Leist M, Nicotera P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ. 1998 Dec;5(12):1001-3. Tahira Farooqui and Akhlaq A. Farooqui. (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385. Tarpley, M.M., D.A. Wink, and M.B. Grisham (2004) Methods for detection of reactive Metabolites of Oxygen and Nitrogen: in vitro and in vivo considerations. Am . J. Physiol Regul Integr Comp Physiol. 286:R431-R444. von Heimburg, D. Hemmrich, K. Zachariah S.,. Staiger, H Pallua, N.(2005) Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells, Respir. Physiol. Neurobiol. 146 (2005) 107–116. Waerzeggers, Yannic Monfared, Parisa Viel, Thomas Winkeler, Alexandra Jacobs, Andreas H. (2010) Mouse models in neurological disorders: Applications of non-invasive imaging, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1802, Issue 10, Pages 819-839. Walker JE, Skehel JM, Buchanan SK. (1995) Structural analysis of NADH: ubiquinone oxidoreductase from bovine heart mitochondria. Methods Enzymol.;260:14–34. Wang A, Costello S, Cockburn M, Zhang X, Bronstein J, Ritz B. (2011). Parkinson’s disease risk from ambient exposure to pesticides. Eur J Epidemiol 26:547-555. Wang, L., Li, J., Li, J., & Liu, Z. (2009). Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1 Wang Y., and Qin ZH., Molecular and cellular mechanisms of excitotoxic neuronal death, Apoptosis, 2010, 15:1382-1402. Wieloch T. (2001). Mitochondrial Involvement in Acute Neurodegeneration 52:247–254. Winklhofer, K. Haass,C (2010) Mitochondrial dysfunction in Parkinson's disease, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1802: 29-44. Yuan, Y., Zheng, J., Zhao, T., Tang, X., & Hu, N. (2016). Uranium-induced rat kidney cell cytotoxicity is mediated by decreased endogenous hydrogen sulfide (H2S) generation involved in reduced Nrf2 levels. Toxicology Research, 5(2), 660-673. doi:10.1039/C5TX00432B Zhang, H., Chang, Z., Mehmood, K., Abbas, R. Z., Nabi, F., Rehman, M. U., . . . Zhou, D. (2018). Nano copper induces apoptosis in PK-15 cells via a mitochondria-mediated pathway. Biological Trace Element Research, 181(1), 62-70. doi:10.1007/s12011-017-1024-0 Zhou, M., Z.Diwu, Panchuk-Voloshina, N. and R.P. Haughland (1997), A Stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: application in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem 253:162-168. AOPWiki 2016-11-29T18:41:23

2022-03-07T07:12:30

Thrombosis and Disseminated Intravascular Coagulation

Thrombosis and DIC

Individual

Thrombosis is defined as the formation or presence of a thrombus. Clotting within a blood vessel may cause infarction of tissues supplied by the vessel. Extreme aggravation of blood coagulation induces multiple thrombi in the microvasculature, which leads to consumption coagulopathy followed by disseminated intravascular coagulation (DIC). DIC is a pathological syndrome resulting from the formation of thrombin, subsequent activation and consumption of coagulation proteins, and the production of fibrin thrombi. The initial pathologic events are thrombotic in nature resulting in thrombotic vascular occlusions.  The initial clinical events are usually hemorrhagic resulting in oozing from mucosa and massive gastrointestinal blood loss. The occlusive events occur as a result of fibrin microthrombi or platelet microthrombi that obstruct the microcirculation of organs. This obstruction can result in organ hypoperfusion and ischemia, infarction, and necrosis. All organs are potentially vulnerable to the effects of thrombotic occlusions. The renal effects of DIC are multifactorial and may be associated with hypovolemia or hypotension. If the hypotension is not corrected it may lead to renal failure due to acute tubular necrosis. Fibrin thrombi may also block glomerular capillaries causing ischemic, renal cortical necrosis (Colman, 1984). The cerebral effects of DIC often result in nonspecific changes such as altered state of consciousness, convulsions, and coma. Major vascular occlusions, subarachnoid hemorrhage, multiple cortical and brain stem hemorrhages may occur following microvascular occlusions (Schwartzman RJ, 1982). The pulmonary effects of DIC may be caused by interstitial hemorrhage resulting in a clinical effect resembling acute respiratory distress syndrome (Schwartzman RJ,1973; Shahl RL, 1984).

Clinical laboratory tests are used to diagnose DIC. Prothrombin time (PT) is a blood test that measures how long it takes blood to clot. PT measures the time required for fibrin clot formation after the addition of tissue thromboplastin and calcium. The average time range for blood to clot is about 10 to 13 seconds. Activated partial prothrombin time (APTT). Platelet poor plasma [PPP] is incubated at 37°C then phospholipid (cephalin) and a contact activator (e.g. Kaolin, micronized silica, or ellagic acid) are added.  This leads to the conversion of Factor XI [FXI] to FXIa. The remainder of the pathway is not activated as no calcium is present.  The addition of calcium (pre-warmed to 37°C) initiates clotting. The APTT is the time taken from the addition of calcium to the formation of a fibrin clot. The clotting time for the APTT lies between 27-35 seconds.   Decreased fibrinogen concentrations <table class="Table" style="background:white; width:100.0%"> <tbody> <tr> <td style="background-color:white; border-bottom:2px solid #dddddd; border-left:none; border-right:none; border-top:none"> Diluted plasma is clotted with a high concentration of Thrombin. The tested plasma is diluted (usually 1:10 but this may vary if the Fibrinogen concentration is very low or very high) to minimize the effect of 'inhibitory substances' within the plasma e.g. heparin, elevated levels of FDPs. The use of a high concentration of Thrombin (typically 100 U/ml) ensures that the clotting times are independent of Thrombin concentration over a wide range of Fibrinogen levels. The test requires a reference plasma with a known Fibrinogen concentration and that has been calibrated against a known international reference standard. A calibration curve is constructed using this reference plasma by preparing a series of dilutions (1:5 –1:40) in the buffer to give a range of Fibrinogen concentrations. The clotting time of each of these dilutions is established (using duplicate samples) and the results (clotting time(s)/Fibrinogen concentration (g/L) are plotted on Log-Log graph paper. The 1:10 concentration is considered to be 100% i.e. normal. There should be a linear correlation between clotting times in the region of 10-50 sec. The test platelet-poor diluted plasma (diluted 1:10 in buffer) is incubated at 37°C, Thrombin [~100 U/mL] added (all pre-warmed to 37°C). The time taken for the clot to form is compared to the calibration curve and the Fibrinogen concentration deduced. Test samples whose clotting times fall out with the linear part of the calibration curve should be re-tested using different dilutions. Most laboratories use an automated method in which clot formation is deemed to have occurred when the optical density of the mixture has exceeded a certain threshold.   Platelet Measurements- A platelet count is the number of platelets a person has per microliter. The ideal platelet range is 150,000 – 400,000 per microliter in most healthy people. Fibrinolysis measurements-         d-dimer concentration ALERE TRIAGE® D-DIMER TEST D-Dimer can be measured by a fluorescence immunoassay. To determine cross-linked fibrin degradation products containing D-dimer in EDTA anticoagulated whole blood and plasma specimens. The test is used as an aid in the assessment and evaluation of patients suspected of having disseminated intravascular coagulation or thromboembolic events including pulmonary embolism Procedure:  Commercially available kits are available to measure d-dimer in whole blood or plasma. The kits contain all the reagents necessary for the quantification of cross-linked fibrin degradation products containing D-dimer in EDTA anticoagulated whole blood or plasma specimens. </td> </tr> </tbody> </table>

Homo sapiens

UBERON:0000178

UBERON blood

Not Specified Unspecific

Not Specified

All life stages

Moderate

Hemostasis and Thrombosis Basic Principles and Clinical Practices Robert W Colman, Jack Hirsh, Victor J. Marder, Edwin W. Salzman (ed) Philadelphia, 1994. Schwartzman RJ, Hill JB: Neurologic complications of DIC. Neurology 32:791, 1982 Robboy SJ, Minna JD, Colman RW et.al. Pulmonary hemorrhage syndrome as a manifestation of DIC: Analysis of 10 cases. Chest 63:718, 1973. Stahl RL, Javid JP, Lackner H: Unrecognized pulmonary embolism presenting as DIC. SM J Med 76:772, 1984. AOPWiki 2021-03-25T19:52:45

2022-11-25T01:38:01

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2021-07-08T10:54:07

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2021-07-08T10:44:50

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eaa49b30> AOPWiki 2021-07-08T10:48:20

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eaa66be0> AOPWiki 2021-07-08T10:50:07

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eaaa3590> AOPWiki 2021-03-30T13:01:13

2021-03-30T13:01:13

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eaafbd80> AOPWiki 2019-07-03T11:52:45

2019-07-03T11:52:45

Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)

TLR9 activation leading to Multi Organ Failure and ARDS

Cataia Ives

BY-SA

2.0

Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.

adjacent

High

High adjacent

Not Specified

Moderate adjacent

High

High adjacent

High

High adjacent

High

High adjacent

High

High adjacent

High

High Mixed

Moderate

Old Age

Not Specified

Adult, reproductively mature

Moderate

Birth to < 1 month

High

Low

High

AOPWiki 2021-03-19T09:45:21

2023-09-25T16:27:05