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

104987-11-3

QJJXYPPXXYFBGM-LFZNUXCKSA-N

Tacrolimus

(Fujimycin) (FK506)

DTXSID5046354

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

D019255

MESH NADPH Oxidase

CHEBI:26523

CHEBI reactive oxygen species

GO:0005739

GO mitochondrion

PR:000000046

PR TGF-beta

GO:0043122

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

GO:0023052

GO signaling

GO:1903409

GO reactive oxygen species biosynthetic process

MP:0003674

MP oxidative stress

MP:0003606

MP kidney failure

WIKI increased

WIKI functional change

Cyclosporin

2017-05-18T08:31:36

Tacrolimus (also FK506)

2016-11-29T18:42:27

2021-06-27T02:54:41

Reactive oxygen species

2017-06-16T08:32:10

2017-08-15T10:43:27

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

Nanoparticles and Micrometer Particles

2022-02-04T13:43:43

9606

NCBI Homo sapiens

WikiUser_28

Vertebrates

WCS_9606

common toxicological species human

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

10090

NCBI mouse

WCS_35525

common ecological species crustaceans

WCS_4472

common ecological species Lemna minor

WCS_7955

common ecological species zebrafish

WikiUser_26

ApacheUser rodents

10116

NCBI rat

WCS_7227

common ecological species Drosophila melanogaster

6239

NCBI Caenorhabditis elegans

Increased 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 (Wink et al 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). NIn accordance with the OECD AOP Handbook, the pathway begins with increased levels of reactive oxygen species (ROS), serving as the Molecular Initiating Event (MIE), which subsequently triggers the Activation of the NF-κB Signaling Pathway . This activation, in turn, directly influences the expression of genes involved in the Differential Production of Cytokines and Chemokines , culminating in the regulation of Pro-Inflammatory Responses Transcriptionally Mediated by NF-κB (. The resultant exaggerated and dysregulated pro-inflammatory response contributes to chronic inflammation and tissue damage, representing the Adverse Outcome (AO). This sequence of events is underpinned by the works of McKay and Cidlowski (1999) and aligns with the guidelines set forth in the OECD AOP Handbook.F-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

McKay LI, Cidlowski JA. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr Rev. 1999 Aug;20(4):435-59. Miller SC, Huang R, Sakamuru S, Shukla SJ, Attene-Ramos MS, Shinn P, Van Leer D, Leister W, Austin CP, Xia M. Identification of known drugs that act as inhibitors of NF-kappaB signaling and their mechanism of action. Biochem Pharmacol. 2010 May 1;79(9):1272-80. Moujalled DM, Cook WD, Lluis JM, Khan NR, Ahmed AU, Callus BA, Vaux DL. In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it. Cell Death Differ. 2012 May;19(5):808-15. Wink S, Hiemstra S, Herpers B, van de Water B. High-content imaging-based BAC-GFP toxicity pathway reporters to assess chemical adversity liabilities. Arch Toxicol. 2017 Mar;91(3):1367-1383. AOPWiki 2016-11-29T18:41:30

2023-08-27T03:08:42

Activation, NADPH Oxidase

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2016-11-29T18:41:30

2017-09-16T10:17:43

Increase, Reactive oxygen species

Increase, ROS

Cellular

Biological State: increased reactive oxygen species (ROS) Biological compartment: an entire cell -- may be cytosolic, may also enter organelles. Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017).  However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015).  <div> Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage. ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. <Free oxygen radicals> <div> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <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:290px"> superoxide </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:290px"> O2·- </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:290px"> hydroxyl radical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ·OH </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:290px"> nitric oxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> NO· </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:290px"> organic radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> R· </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:290px"> peroxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROO· </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:290px"> alkoxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RO· </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:290px"> thiyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RS· </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:290px"> sulfonyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROS· </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:290px"> thiyl peroxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RSOO· </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:290px"> disulfides </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RSSR </td> </tr> </tbody> </table> </div> <Non-radical ROS> <div> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <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:290px"> hydrogen peroxide </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:290px"> H2O2 </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:290px"> singlet oxygen </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> 1O2 </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:290px"> ozone/trioxygen </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O3 </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:290px"> organic hydroperoxides </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROOH </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:290px"> hypochlorite </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ClO- </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:290px"> peroxynitrite </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ONOO- </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:290px"> nitrosoperoxycarbonate anion </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O=NOOCO2- </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:290px"> nitrocarbonate anion </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O2NOCO2- </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:290px"> dinitrogen dioxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> N2O2 </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:290px"> nitronium </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> NO2+ </td> </tr> <tr> <td colspan="2" style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:580px"> highly reactive lipid- or carbohydrate-derived carbonyl compounds </td> </tr> </tbody> </table> </div> Potential sources of ROS include NADPH oxidase, xanthine oxidase, mitochondria, nitric oxide synthase, cytochrome P450, lipoxygenase/cyclooxygenase, and monoamine oxidase [Granger et al., 2015]. ROS are generated through NADPH oxidases consisting of p47phox and p67phox. ROS are generated through xanthine oxidase activation in sepsis [Ramos et al., 2018]. Arsenic produces ROS [Zhang et al., 2011]. Mitochondria-targeted paraquat and metformin mediate ROS production [Chowdhury et al., 2020]. ROS are generated by bleomycin [Lu et al., 2010]. Radiation induces dose-dependent ROS production [Ji et al., 2019]. ROS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation [Dickinson and Chang 2011; Egea et al. 2017]. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide (NO) is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. NO production is one of the main mechanisms by which phagocytes kill bacteria [Wang et al., 2017]. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes. ROS activity is principally local. Most ROS have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrite can survive long enough to diffuse across membranes [Calcerrada et al. 2011]. Consequently, local concentrations of ROS are much higher than average cellular concentrations, and signaling is typically controlled by colocalization with redox buffers [Dickinson and Chang 2011; Egea et al. 2017]. Although their existence is limited temporally and spatially, ROS interact with other ROS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase RNS. Both ROS and RNS also move into neighboring cells, and ROS can increase intracellular ROS signaling in neighboring cells [Egea et al. 2017]. In the primary event, photoreactive chemicals are excited by the absorption of photon energy.  The energy of the photoactivated chemicals transfer to oxygen and then generates the reactive oxygen species (ROS), including superoxide (O2−) via type I reaction and singlet oxygen (1O2) via type II reaction, as principal intermediate species in phototoxic reaction (Foote, 1991, Onoue et al. , 2009). </div>

Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies. Yuan, Yan, et al., (2013) described ROS monitoring by using H2-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H2-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm. Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013). Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS. On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006).  The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014). <div> <Direct detection> Many fluorescent compounds can be used to detect ROS, some of which are specific, and others are less specific. ・ROS can be detected by fluorescent probes such as p-methoxy-phenol derivative [Ashoka et al., 2020]. ・Chemiluminescence analysis can detect the superoxide, where some probes have a wider range for detecting hydroxyl radical, hydrogen peroxide, and peroxynitrite [Fuloria et al., 2021]. ・ROS in the blood can be detected using superparamagnetic iron oxide nanoparticles (SPION)-based biosensor [Lee et al., 2020]. ・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. ・The levels of ROS can be quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference node [Flaherty et al., 2017]. ・Singlet oxygen can be measured by monitoring the bleaching of p-nitrosodimethylaniline at 440 nm using a spectrophotometer with imidazole as a selective acceptor of singlet oxygen [Onoue et al., 2014]. <Indirect Detection> Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione [Dickinson and Chang 2011; Wang et al. 2013; Griendling et al. 2016]. However, these methods cannot generally distinguish between the oxidative species behind the changes and cannot provide good resolution for the kinetics of oxidative activity. </div>

ROS is a normal constituent found in all organisms, lifestages, and sexes.

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific High Mixed

High

All life stages

High Moderate Moderate Moderate High High High

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2025-06-12T01:27:08

Increase, Oxidative Stress

Molecular

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

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

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

High Mixed

High

All life stages

High High

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(2016), “Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association”, Circulation research, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/RES.0000000000000110" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/RES.0000000000000110</a>   Guo, C. et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, Neural regeneration research, Vol. 8/21, Publishing House of Neural Regeneration Research, China, <a href="https://doi.org/10.3969/j.issn.1673-5374.2013.21.009" rel="noreferrer noopener" target="_blank">https://doi.org/10.3969/j.issn.1673-5374.2013.21.009</a>  Hargreaves, M., and L. L. 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(2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, International journal of molecular medicine, Vol. 44/1, Spandidos Publishing Ltd., Athens, <a href="https://doi.org/10.3892/ijmm.2019.4188" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm.2019.4188</a>  AOPWiki 2017-05-30T13:58:17

2026-02-11T07:05:27

Increase, Mitochondrial dysfunction

Cellular

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

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

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

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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. Orrenius, S., Gogvadze, V., & Zhivotovsky, B. (2015). Calcium and mitochondria in the regulation of cell death. Biochemical and Biophysical Research Communications, 460(1), 72-81. doi:10.1016/j.bbrc.2015.01.137 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. Plitzko B, Loesgen S. Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism. Bio Protoc. 2018 May 20;8(10):e2850. doi: 10.21769/BioProtoc.2850. PMID: 34285967; PMCID: PMC8275291.  Pourahmad, J., Ghashang, M., Ettehadi, H. A., & Ghalandari, R. (2006). A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity. Environmental Toxicology, 21(4), 349-354. doi:10.1002/tox.20196 Pozzan, T., & Rudolf, R. (2009). 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Calcineurin inhibitor induced nephrotoxicity leading to kidney failure

Calcineurin inhibitor induced nephrotoxicity

Brendan Ferreri-Hanberry

K.H. Liang, Department of Nephrology, University Medical Center Utrecht E. Sendino Garví, Department of Pharmaceutical Sciences, Utrecht University A.M. van Genderen, Department of Pharmaceutical Sciences, Utrecht University R. Masereeuw, Department of Pharmaceutical Sciences, Utrecht University

BY-SA

2.7

Tacrolimus (TAC) and cyclosporin A (CsA) are widely used calcineurin inhibitors (CNI) whose therapeutic efficacy (i.e. immunosuppression) is limited by nephrotoxicity, particularly in kidney transplant recipients. This adverse outcome pathway (AOP) outlines the mechanistic sequence of events leading from CsA or TAC exposure to chronic kidney injury. Despite the molecular initiating event remains unknown, the first recorded key event is the activation of nuclear factor kappa B (NF-κB) and induction of NADPH oxidase 2 (NOX2). These upstream events promote increased production of reactive oxygen species (ROS), a well-established response observed in vitro and in vivo. Elevated ROS levels drive oxidative stress, characterized by depletion of antioxidant capacity and increased oxidative damage markers. Oxidative stress leads to mitochondrial dysfunction, including loss of membrane potential, impaired respiration, and reduced ATP production. These mitochondrial changes are accompanied by metabolic disturbances, such as alterations in TCA cycle intermediates, amino acid pathways, and fatty acid metabolism, described across cell-based systems and in vivo studies. Metabolic impairment and cellular stress are associated with increased TGF-β signalling, a central event consistently linked to CNI-induced tubular injury and fibrosis. Persistent activation of TGF-β pathways contributes to extracellular matrix deposition, tubular atrophy, and interstitial fibrosis. These tissue-level alterations ultimately culminate in decreased kidney function, reflected clinically by reduced GFR and progression toward chronic kidney disease. This AOP provides a structured framework to integrate mechanistic evidence, identify key data gaps, and support the development of  new approach methodology-based approaches for assessing CNI nephrotoxicity.   This AOP describes the sequence of key events (KE’s) that link the usage of CNIs to nephrotoxicity and the adverse outcome kidney failure. CNIs are immunosuppressive drugs that inhibit calcineurin by binding to immunophilins, such as cyclophilin and FK-binding proteins (1). Calcinuerin is a crucial enzyme for activating T-cells. Blockage of the enzyme leads to reduction of interleukin-2 and T-cell proliferation. Currently, the two most commonly used systemic CNIs are TAC and CsA, typically administered  orally. Indication for systemic CNI treatment is solid organ transplantation and are used to effectively manage several autoimmune disorders, including lupus nephritis, idiopathic inflammatory myositis and interstitial lung disease. CNIs are associated with both acute and chronic kidney damage due to its narrow therapeutic window (2, 3). Nephrotoxicity secondary to CNIs occurs in liver (52%), heart (20–75%) and kidney (76–94%) transplant recipients (4). Risk factors for CNI toxicity include volume depletion, diuretic use, older donor age, exposure to high CNI doses, concomitant use of nephrotoxic drugs (particularly NSAIDs), concomitant use of CYP3A4/5 or P-glycoprotein (ABCB1) inhibitors, and genetic polymorphisms affecting CYP3A4/5 and P-glycoprotein function. CNI are also available in a topical form, which is used for mild-to-moderate atopic dermatitis. However, because of the route of exposure, nephrotoxicity is not an issue for topical CNIs.     <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol> <li style="text-align:justify">Molecular Initiating Event: Unkown</li> </ol> </li> </ol> </li> </ol> As CNI-induced nephrotoxicity involves several mechanisms, a well-defined MIE cannot be found. CNI-induced nephrotoxicity can be a result of vasoconstriction of the afferent arteriole, leading to ischemia in the kidney (2). However, there is also a direct toxic effect, such as increased oxidative stress, of CNI’s on tubular epithelial cells, as has been shown in multiple studies (5-7). In tubular epithelial cells, the influx transporter, if existing, of CNI’s is unknown, and the only efflux transporter that has been identified to transport CNI’s is P-glycoprotein (ABCB1). Identifying the initial interaction of CNI with kidney cells, and consequently the MIE, remains challenging. <h3 style="text-align:justify"> </h3> <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="2"> <li style="text-align:justify">Key Event 1: Activation of NF-kB and NOX2 </li> </ol> </li> </ol> </li> </ol> NF-κB, a downstream target of CNI, is activated in tubular cells after treatment with TAC and CsA (8). The pathway of NF-κB activation is not exactly known, but it could be activated by angiotensin II (9), degradation of I-κBα (10) or upstream kinases: c-Jun N-terminal kinases (JNK) and Janus Kinase (JAK)(11). A transcriptomics analysis of mouse cortical proximal tubular cells treated with TAC and CsA showed significant activation of NF-κB and pro-inflammatory cytokines which are strongly associated with kidney disease, such as monocyte chemoattractant protein -1 (MCP-1) and Rantes (11). Inhibition of Nf-κB after CNI exposure in several in vivo studies have shown attenuation of nephropathy, indicating an important role of the factor in the development of CNI nephrotoxicity (12, 13). Additionaly, activation of NF-κB leads to upregulation of NADPH oxidases (NOX) in the tubule (14).   The primary function of NOX are generating reactive oxidative species (ROS) by transferring one electron to dioxygen leading to the product superoxide. Isoforms NOX1, NOX2 and NOX4 are expressed in the kidney and known sources of ROS production (15).  NOX2 in particular is associated with CsA-induced kidney fibrosis and chronic nephrotoxicity (16, 17). Furthermore, NOX2 was significantly increased in the tubule and interstitial cells after immunohistochemical analyses of kidney biopsies from liver transplant recipients with CNI nephrotoxicity after exposure to TAC or CsA (16). Moreover, TAC increases ROS via NOX in the glomerulus, especially in endothelial glomerular cells (18).   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="3"> <li style="text-align:justify">Key Event 2: ROS production</li> </ol> </li> </ol> </li> </ol> Mitochondria generate about 90% of cellular ROS, making them the primary source of ROS in renal proximal tubules (19). The kidney is highly metabolically active, abundant in mitochondira, and, therefore, particularly susceptible to ROS. When ROS production exceeds the cell’s antioxidant capacity, oxidative stress occurs, damaging cellular components and contributing to chronic kidney disease (CKD) progression (20). In a proximal tubular cell line derived from a healthy human adult male kidney (human kidney-2 (HK-2) cells), TAC increased the intracellular ROS levels by 1.67 fold compared to the control group (21). Exposure to TAC (30 µM and 60 µM, for 24 h) in induced pluripotent stem cell (iPSC) derived  kidney organoids significantly increased ROS levels detected by immunofluorescence staining with MitoSOX Red (22). Moreover, damaged mitochondria with spherical shapes and cristolysis were observed in the kidney organoids after TAC exposure, and quantification with Mitrotracker showed increased mitochondrial stress (22).     In addition to increasing ROS, CNI also disrupt antioxidant defense systems leading to imbalances in the redox environment, amongst which decreasing glutathione (23). Moreover, CsA exposure damages the inner and outer mitochondrial membrane, resulting in mitochondrial permeability transition pores. The exact mechanism has yet to be elucidated, but it has been suggested that oxidative stress may alter thiol groups of membrane proteins, leading to misfolding and clustering of these proteins and resulting in opening of membrane pores (23).  Subsequently, apoptotic mediators, such as cytochrome c, are released into the cytosol. Moreover, increased ROS might lead to increased expression of dynamin related protein 1, a GTPase that regulates mitochondrial fission and decreased expression of mitofusin 2  and optic atrophy protein 1 (Opa1), both important proteins in the mitochondrial fusion process (24, 25). Consequently, increased fission and decreased fusion takes place in the mitochondria, dysregulating the apoptotic pathways (26, 27).   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="4"> <li style="text-align:justify">Key Event 3: Oxidative stress </li> </ol> </li> </ol> </li> </ol> Oxidative stress, defined by an imbalance between pro-oxidant and antioxidant systems, is harmful to cellular health due to the excessive production of ROS and reactive nitrogen species  (28). Evidence strongly indicates this to be a KE in TAC-induced nephrotoxicity (7, 29-31). A study in (porcine-derived) LLC-PK1 cells demonstrated an increase in hydrogen peroxide, a direct indicator of oxidative stress upon TAC treatment with increased expression of pro-oxidant enzymes and reduced activity of antioxidant pathways, resulting in cellular damage and apoptosis (7). In vitro studies using kidney cell lines and kidney organoids offer deeper insights into the cellular and molecular mechanisms of TAC-induced oxidative stress. Kidney proximal tubular epithelial cells exposed to TAC exhibit upregulation of oxidative stress markers and mitochondrial dysfunction (32). These findings are supported by several other studies, which reported downregulation of antioxidants and upregulation of NOX2 after TAC in vitro and in vivo. Furthermore, treatment with coenzyme Q10 has been shown to alleviate TAC-induced kidney dysfunction by preventing ROS production and improving mitochondrial respiration in HK-2 cells (33). In vivo studies in rodents further clarify the mechanisms by which TAC induces oxidative stress (34). Further, in an intracellular metabolomics analyses, mice treated with CNIs showed increased oxidative stress by either decreased glutathione or decreased glutathione peroxidase activity in the kidney cells (7). Rabbits exposed to TAC showed increased oxidative stress caused by impairments in antioxidant response (35). Additionally, the anti-oxidant cilastatin counteracted TAC-induced nephrotoxicity in rat proximal tubular cells, resulting in reduced oxidative DNA damage (33). Overall, the use of antioxidants and oxidative stress inhibitors has shown potential in mitigating the oxidative damage caused by TAC in vitro and in vivo, suggesting possible therapeutic strategies to counteract its nephrotoxic  effects (36). Human studies have also indicated that patients undergoing TAC therapy exhibit decreased antioxidant defenses (37, 38) including reduced glutathione levels. Additionally, increased levels of malondialdehyde, indicating a process called lipid peroxidation, as a result of oxidative stress have been found in patients. Collectively, these findings underscore oxidative stress as an early key event in TAC exposure, potentially initiating the cascade of events that eventually leads to kidney failure.   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="5"> <li style="text-align:justify">Key Event 4: Mitochondrial dysfunction</li> </ol> </li> </ol> </li> </ol> The kidney, with its high energy demands for reabsorption processes across different nephron segments, relies heavily on mitochondria. Mitochondrial dysfunction is a significant factor, leading to impaired ATP production, increased ROS levels, metabolic deficiencies and damage that directly impact kidney function (39). In vitro studies using HK-2 cells have shown that TAC exposure causes a loss of mitochondrial membrane potential. These effects were accompanied by decreased oxygen consumption rates and impaired ATP synthesis, further confirming mitochondrial dysfunction (33). Also kidney organoids treated with TAC exhibit significant mitochondrial damage, including loss of mitochondrial membrane potential, increased ROS production, and activation of autophagy as a cellular response to mitochondrial stress (40). Additionally, coenzyme Q10, a mitochondrial-targeted antioxidant, could partly mitigate the mitochondrial dysfunction and oxidative stress induced by TAC (41). In vivo studies have shown that TAC administration in mice results in increased mitochondrial ROS production and decreased antioxidant defenses, exacerbating mitochondrial dysfunction and kidney injury (42). Electron microscopy of kidney tissues from TAC-treated mice revealed a reduction in both the number and volume of mitochondria, along with structural abnormalities (33), which was also demonstrated in rats, with the remaining mitochondria appearing fragmented (fission and fusion) (33). These findings suggest that TAC not only impairs mitochondrial function but also affects mitochondrial biogenesis and morphology. Long-term exposure of human patients to TAC leads to progressive kidney failure, characterized by interstitial fibrosis, tubular atrophy, and inflammation (43), all of this potentially linked to mitochondrial damage. In agreement, ultrastructural analysis of kidney biopsies from patients treated with TAC revealed mitochondrial swelling, cristae disruption, and increased ROS production (44).   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="6"> <li style="text-align:justify">Key Event 5: Metabolic impairment </li> </ol> </li> </ol> </li> </ol> Cell metabolism impairment is a critical event in the pathway of TAC-induced nephrotoxicity, encompassing various disruptions that contribute to kidney damage and dysfunction (42, 45). Within mitochondria, the TCA cycle, fueled by glycolysis-derived pyruvate, produces energy-rich nucleotides and accumulates coenzyme NADH+ in the presence of oxygen (46, 47). NADH is processed by the mitochondrial respiration chain complex (subunits I-IV), along with cytochrome C and coenzyme Q10, creating a gradient that converts ADP into ATP, which is then transported into the cytosol (48). In vivo studies have provided additional insights into the mechanisms of TAC-induced nephrotoxicity. For instance, in a mouse model, TAC administration resulted in significant alterations in kidney metabolism, including changes in metabolites such as L-valine and D-glucose, indicative of disrupted mitochondrial function (42, 45). Treatment with non-toxic doses of TAC showed impairments in the TCA cycle, including increased folate metabolism, citric acid metabolism, and glutathione synthesis, indicating a metabolic switch as a reaction to oxidative stress (G) (42, 45). In proximal tubule cells, L-carnitines were the most differentially accumulated metabolites upon TAC exposure in. Other in vitro studies using kidney cell lines have also demonstrated the impact of TAC on cellular metabolism including mitochondrial function disruption leading to decreased ATP production and increased ROS production (49, 50). These metabolic changes induce cellular stress and apoptosis, contributing to tubular dysfunction and fibrosis. Additionally, they found that TAC inhibits key metabolic enzymes, such as pyruvate dehydrogenase and citrate synthase, further impairing cellular energy metabolism. These findings highlight the direct effects of TAC on kidney cell metabolism and its role in nephrotoxicity development. Furthermore, another study found TAC-induced metabolic impairment involved alterations in amino acid metabolism, with decreased levels of essential amino acids such as valine and leucine (7). These changes impair protein synthesis and cellular repair mechanisms, further exacerbating kidney damage. In clinical settings, TAC-induced nephrotoxicity is often observed in kidney transplant recipients (50). Studies have shown that TAC can cause metabolic disturbances, including hyperglycemia and dyslipidemia, which are risk factors for CKD (51). TAC impairs glucose metabolism by reducing insulin secretion and increasing insulin resistance, leading to hyperglycemia (52). Additionally, the same study found TAC linked to alterations in lipid metabolism, resulting in elevated levels of cholesterol and triglycerides. These metabolic changes exacerbate kidney injury by promoting oxidative stress and inflammation within the kidney.   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="7"> <li style="text-align:justify">Key Event 6: Aberrant TGF-βeta</li> </ol> </li> </ol> </li> </ol> Exposure to TAC or CsA has been associated with significant alterations in TGF-β expression in the kidney, contributing to cytoxicity and fibrosis (53). An increased TGF-β expression has been observed with an aberrant activation of the TGF-β receptor stimulating the smad-mediated production of extracellular matrix (ECM) components, including collagen and fibronectin, features of fibrosis (49, 54). In transplant recipients, long-term treatment with TAC or CsA resulted in elevated intrarenal expression of TGF-β, collagen, fibronectin, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinases-2 (TIMP-2), and osteopontin, with TAC showing more pronounced effects than CsA (53). The TGF-β-inducible gene-H3 is a product induced by TGF-β1 and plays a role in the fibrotic response. In vitro studies have demonstrated that TAC induces fibroblast-to-myofibroblast transition via a TGF-β-dependent mechanism, which is an indication of fibrosis (55). This process involves the inhibition of the calcineurin (Cn)/nuclear factor of activated T cells (NFAT) axis, induction of TGF-β1 ligand secretion, and receptor activation in kidney fibroblasts. Furthermore, anti-TGF-β treatment in animal models has been shown to prevent nephrotoxicity induced by CNIs, highlighting the therapeutic potential of targeting the TGF-β pathway to mitigate kidney fibrosis (56).   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="8"> <li style="text-align:justify">Key Event 7: Increase, kidney fibrosis</li> </ol> </li> </ol> </li> </ol> TAC-treated kidneys exhibit significant evidence of kidney fibrosis, including increased expression of alpha-smooth muscle actin (α-SMA), indicating fibroblast activation (55, 57). Nitric oxide modulation has been proposed to affect fibrosis by altering TGF-β1 expression, leading to increased matrix deposition and decreased matrix degradation through increased plasminogen activator inhibitor-1 (58). In addition to increased TGF-β expression, macrophage infiltration and cellular proliferation are critical cellular players contributing to tubulointerstitial fibrosis (59). Cellular proliferation occurs both in the tubular and interstitial regions, starting in the medulla and progressing to fibrotic areas. Macrophages, which produce pro-fibrotic cytokines such as TGF-β and platelet-derived growth factor, may infiltrate during the early phases of fibrosis (60). This infiltration is potentially related to increased expression of osteopontin (OPN), a chemoattractant for macrophages, following CsA exposure (61). In vitro studies on PTECs have shown that both TAC and CsA induce significant OPN mRNA expression (62). Treatment of mice with TGF-β resulted in increased intra-renal OPN mRNA and protein expression. In TAC-treated animals, a parallel increase in OPN and TGF-β mRNA was observed. Anti-TGF-β antibody treatment in vitro inhibited both TGF-β and OPN mRNA expression in PTECs, and similar results were obtained in vivo with CsA-treated mice (63). In support, OPN-deficient mice exhibit less severe CsA nephrotoxicity, characterized by reduced interstitial collagen deposition and macrophage infiltration, compared to control mice (61). Macrophage influx has been correlated with apoptosis in kidney tissue of CsA-treated rats (64). Similarly, OPN expression has been associated with increased microvascular injury in CsA nephrotoxicity, suggesting it could be an early marker of CNI toxicity (65).   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="9"> <li style="text-align:justify">Key Event 8: Apoptosis </li> </ol> </li> </ol> </li> </ol> TAC and CsA have been directly related to cellular damage that activate apoptotic pathways (5, 6), which is primarily driven by oxidative stress and direct toxic effect on tubular cells (7). Additionally, TAC can activate the Fas system, a critical pathway in apoptosis, leading to increased cell death in kidney tissues (66). Treatment with TAC and CsA activates the endoplasmic reticulum (ER) stress pathway, leading to the upregulation of CHOP (C/EBP homologous protein), a key mediator of ER stress-induced apoptosis (67). These cellular events culminate in the activation of caspase-12 and caspase-3 (68), executing the apoptotic process. Furthermore, in a study involving mice, TAC administration led to tubular atrophy and interstitial fibrosis, with a marked increase in apoptotic cells in the kidney cortex (66). In the clinic, biopsies from kidney transplant recipients on TAC therapy revealed increased apoptotic markers, such as caspase-3 activation and DNA fragmentation (64). These findings suggest that TAC-induced apoptosis contributes to the deterioration of kidney function in humans.   <ol> <li style="list-style-type:none"> <ol> <li style="list-style-type:none"> <ol start="10"> <li style="text-align:justify">Adverse Outcome: Kidney failure</li> </ol> </li> </ol> </li> </ol> Kidney biopsies taken from patients exposed to TAC and CsA revealed several clinical manifestations of nephrotoxicity. These include mild arteriolopathy, striped interstitial fibrosis, glomerular congestion, tubular microcalcification and arterial hyalinosis (69). Other than histological abnormalities, decline in kidney function is often observed in patients (70). Kidney function can be estimated by measuring serum creatinine levels. Creatinine is a waste product in the body that is freely filtered at a constant rate and minimally reabsorbed by the kidney. Using serum creatinine, the estimated glomerular filtration rate (eGFR) can be calculated with a formula that adjusts for age and sex. Kidney failure, e.g. end-stage kidney disease, is defined as an eGFR below 15 mL/min/1.73 m². Next to declined eGFR, other indicators for declined kidney function are decreases in urea clearance, leading to increased blood urea nitrogen (70). Urea is the end product of protein metabolism formed in the liver and is excreted, reabsorbed and transported by the kidneys. Studies have reported that 9.5% to 16.5% of patients develop kidney failure as a result of continuous CNI usage (71, 72).

This adverse outcome pathway (AOP) is developed as part of the ‘Virtual Human for Safety Assessment (VHP4Safety)’ consortium. The mission of the consortium is to improve the prediction of the potential harmful effects of chemicals and pharmaceuticals based on a holistic, interdisciplinary definition of human health by developing the Virtual Human Platform and accelerating the transition from animal-based testing to innovative safety assessment. The Virtual Human Platform integrates data on human physiology, chemical characteristics and perturbations of biological pathways, for the first time in an inclusive and integrated manner. This project is funded by the Dutch Research Council (NWO) programme entitled the ’Dutch Research Agenda: Research on Routes by Consortia (NWA-ORC). Within the VHP consortium, in vitro case studies are used to feed the Virtual Human Platform with newly generated data. This AOP focuses on nephrotoxicity caused by tacrolimus (TAC), cyclosporin A (CsA) and other calcineurin inhibitors leading to kidney failure.

Not Specified Unspecific

Not Specified

Not Otherwise Specified

High High Moderate

In this section, we assessed the AOP using the Brasdford-Hill criteria (73), including the AOP’s biological plausibility and assessment of the empirical evidence: dose-response and temporal concordance. We also assessed the overall KEs within the AOP, including their domain of applicability and essenciality.   Biological Plausibility Currently, there is no evidence supporting the intracellular transport of calcineurin inhibitors, specifically TAC and CsA. TAC has been documented to be secreted from proximal tubule cells in the kidney via the P-glycoprotein efflux pump, encoded by the ABCB1/MDR1 gene (74). However, it remains unclear whether this transport is exclusively mediated by P-glycoprotein or involves other transporters. This gap in knowledge regarding the cellular transport mechanisms of these agents presents a significant challenge in elucidating the initial triggering event and subsequent KEs and relationships (KERs). Although the MIE could not be identified, it has been suggested that NF-κB and NOX2 activation are among the earliest key events in nephrotoxicity induced by CNI (14, 16, 17). Exposure to these nephrotoxic agents results in increased expression of both NF-κB and NOX2. The activation of NF-κB and NOX2 promotes the generation of ROS by transferring an electron from NADPH to molecular oxygen within the mitochondrial respiratory chain. In this AOP, although the sequence of KEs is depicted linearly, we propose that the KEs of ROS production, oxidative stress, and mitochondrial dysfunction are fundamentally interconnected and dynamic processes that occur simultaneously. This simultaneity complicates the assessment of whether one event precedes another or if they occur concurrently. Given that critical metabolic processes occur in the mitochondria, mitochondrial dysfunction results in metabolic impairment (7). Studies have identified the metabolic pathways of arginine, amino acids, and pyrimidine as the most affected by TAC exposure in both in vitro and in vivo models (7). Metabolic impairment due to CNI exposure has been associated with aberrant TGF-β signaling and subsequent fibrosis in kidney cells and animal models (49). Evidence suggests that fibrosis may arise from either increased TGF-β levels or malfunctions in the TGF-β receptor signaling pathway, or a combination of both (49). Increased fibrosis has been observed alongside the presence of pro-apoptotic and apoptotic cells in in vitro, animal, and human studies (64, 75). However, apoptosis can be triggered by various stressors, including oxidative stress, mitochondrial dysfunction, and increased fibrosis, complicating its placement within the AOP framework. Ultimately, increased apoptosis leads to the loss of functional kidney tissue and eventual kidney failure.   Empirical evidence: Dose-response and temporal concordance CNI induced kidney damage in patients is associated with the dosage of CNI. A mean reduction in TAC dosage of 41% (range 11–89) led to a 86% (range 45–100) reduction in serum creatinine within 1–14 days (76). To decrease CNI usage, combining CNI with or switching to other immunosuppressive drugs seems to be able to partly restore kidney function. Switching from CNI to sirolimus for example led to an increase of 27% of eGFR (from 34 to 42 ml/min/1.73m2) in adult liver transplant recipients (77). Adding immunosuppressant mycophenolate mofetil to reduce CNI doses led to decreased serum creatinine levels from 2.63+/-0.39 to 1.74+/-0.34 mg/dl after one month and was maintained within a follow-up period of 4.8 years in adult liver transplant recipients (78). In the pediatric heart transplant population, improvement of kidney function after decreasing CNI by 50% and adding mycophenolate was also observed. At 1 year post-intervention, GFR was increased by 67% from 46.5 to 77.6mL/min/1.73 m2a nd remained stable during the mean follow-up of 26.3 months (79). In animal models, male Sprague-Dawley rats dosed daily with CsA (2.5 or 25 mg/kg/day), TAC (0.6 or 6 mg/kg/day) for 1-28 days, a significant increase in blood urea nitrogen was observed in rats treated with CsA (high dose) or TAC (high dose) for 14 and 28 days (80). In another study, significant impairment of eGFR was seen in Sprague-Dawley rats treated with doses of CsA as low as 5 mg/kg/day (81). CsA 7.5 mg/kg/day caused a significant reduction in effective kidney plasma flow, and at 10 mg/kg/day filtration fraction declined significantly, again, implying that CNI-induced kidney failure is dose-dependent (81). In vitro models also showed that several KE’s in this AOP are dose-dependent. First of all, Jin et al. showed a dose-response in cell viability when HK-2 cells were exposed to TAC (82). Additionally, increased ROS production and acecelerated apoptosis were found in those cells in a dose-dependent manner. In human mesangial cells, CsA exposure led to a dose-dependent loss of viability, and only after 48 hours a decrease in cell proliferation was observed, suggesting that CsA nephrotoxicity is also time-dependent (83). In PTEC, the oxygen consumption rate and activity of the electron transport chain complexes I, II, IV were dose-dependently decreased after 48 hour exposure to CsA whereas glycolysis, measured by the extracellular acidification rate is dose-dependently increased, which both could be an indicator of mitochondrial dysfunction (84). In conclusion, aforementioned in vitro, in vivo and in clinical results imply that CNI treatment is associated with several KE’s and kidney failure in a dose- and time-dependent manner. Mechanistic evidence for KEs and KERs in this AOP primarily comes from rodent studies and immortalized kidney cell lines, and limited clinical evidence. Due to this, it remains unclear whether this AOP can be fully applied to other species and life stages. This AOP outlines the general mechanisms leading to kidney failure across various species, including humans and rodents. Theoretically, this AOP could be applicable to all life stages and any organism capable of experiencing CNI-related kidney failure. There is an edivent limitation in empirical support, with very limited studies detailing the KEs and KERs. Additionally, there is limited information on the dose- and time-dependent response relationships for most of the stressors within this AOP, particularly in studies that measure the relationship between the KEs in the context of CNI exposure and the kidney. Therefore, additional quantitative data is needed before this AOP can be considered for regulatory significance.

<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; margin-left:-38px; width:680px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:32px; width:66px"> MIE/KE </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:32px; width:92px"> Short name </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:32px; width:354px"> Support </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:32px; width:168px"> Essentiality </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> MIE </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> N/A </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> Unidentified event </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> N/A </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> NF-κB and NOX2 activation </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> NF-κB inhibition by affeic acid phenethyl ester suppressed NOX2 protein expression in both WT and CnAα−/− kidney fibroblasts, and significantly reduced ROS levels in CnAα−/− cells, indicating that NF-κB mediates CnAα-induced NOX2 regulation (14). Inhibition of NOX2 by coadministration of apocynin or diphenyleneiodonium in Fisher344 rats treated with CsA was associated with reduced striped fibrosis and cytoplasmic vacuolization that characterize chronic CNI toxicity (16). Additionally, treatment of wild-type and Nox2 null (B6.129S-CybbTm1Din/J) mice with high dose CsA revealed that in the KO mice significantly lower levels of α-SMA and 4-hydroxynonenal protein were found in the kidney, indicating that NOX2 is a mediator in chronic CNI toxicity. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> High </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 2 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> ROS increase </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> CTLA4-Ig, a fusion protein, can effectively reduce TAC-induced increase in ROS levels and oxidative stress in HK-cells. Additionally, in rats, CTLA4-Ig treatment significantly reversed kidney function indices induced by TAC, including blood urea nitrogen, creatinine, malondialdehyde, glutathione, 8-OHdG, 4-HHE, catalase, glutathione S-transferase and glutathione reductase(82). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> High </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 3 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> Oxidative stress increase </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> Treatment with coenzyme Q10 has been shown to reduce mitochondrial ROS production and alleviate TAC-induced dysfunction in the kidney by preventing apoptosis in HK-2 cells(31). Additionally, treatment with cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig) has been reported to alleviate TAC induced oxidative stress and apoptotic cell death in HK-2 cells via the activation of the protein kinase B (AKT)/forkhead transcription factor (FOXO) 3 pathway (82). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> High </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 4 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> Mitochondrial dysfunction </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> HK-2 cells treated with TAC and coenzyme Q10, an antioxidant, had higher rates of basal mitochondrial respiration than those treated with TAC only, indicating an increased activity of mitochondria. Additionally, the cells treated with coenzyme Q10 also showed higher ATP-associated and total respiration (31). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> Low/Medium </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 5 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> Metabolic impairment </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> A study showed that CTLA4-Ig treatment in mice and HK-2 cells exposed to TAC significantly reversed the altered levels of several key metabolites, including blood urea nitrogen, creatinine, malondialdehyde, glutathione, 8-OHdG, 4-HHE, catalase, glutathione S-transferase, and glutathione reductase (82). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> Low/Medium </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 6 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> Aberrant TGF-β </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> In vitro studies using conditionally immortalized PTECs and in vivo studies have demonstrated that inhibition of TGF-β prevented TGF-β activation and consequent fibrosis signalling in the context of Cas exposure (56, 85).   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> Low/Medium </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 7 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> Increased kidney fibrosis </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> In vivo treatment with TGF-β in mice results in increased intra-renal osteopontin (OPN) mRNA and protein expression (85). In TAC-treated animals, a parallel increase in OPN and TGF-β mRNA was observed. Fibrosis inhibition by TGF-β blockage downregulated both TGF-β and OPN mRNA expression in PTECs, with similar results obtained in vivo with CsA-treated mice (86), leading to macrophage infiltration and consequent inflammation and fibrosis.   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> Low/Medium </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:30px; width:66px"> 8 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:92px"> Apoptosis </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:354px"> Treatment with coenzyme Q10 was shown to alleviate TAC-induced dysfunction in the kidney by preventing apoptosis in HK-2 proximal tubule cells (31). Additionally, cilastatin treatment acts as an anti-apoptotic agent in TAC-induced nephrotoxicity in proximal tubular cells and in rat models (87). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:30px; width:168px"> High </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:32px; width:66px"> AO </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:32px; width:92px"> Kidney failure </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:32px; width:354px"> CNI treatment, including mostly TAC and CsA, has been associated with both acute and CKD due to its narrow therapeutic window (2, 3). Nephrotoxicity due to treatment with CNIs occurs often in solid-organ transplantation, including liver (52%), heart (20–75%) and kidney (76–94%) transplant recipients (4). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:32px; width:168px"> High </td> </tr> </tbody> </table>

The clinical evidence, linking nephrotoxicity caused by CNI to kidney failure, is strong and reliable. However, the mechanistic links between exposure to the agents and kidney failure remain poorly described and, therefore, not well understood. We were unable to find any evidence of a specific MIE, which thus remains unknown. The toxicity trigger of CNIs can be influenced by the kinetics of the individual stressors, which are determined by the transporters that these agents use to enter cells. Except for P-glycoprotein, neither influx nor efflux transporters have been identified, which hampers the identification of the MIE and the potential subsequent cascade of events. Currently, there are no reported alternative mechanisms that deviate from the proposed AOP, but additional contributors to the current AOP should not be excluded. This AOP is a qualitative description of the pathway and does currently not include quantitative information on dose-response relationships. However, it is known that CNI treatment needs frequent drug monitoring due to its narrow therapeutic window. There is substantial inter-individual variability in drug exposure, which may result from differences in metabolic enzymes (CYP3A4/5) and transporter polymorphisms (ABCB1), as well as the impact of high-fat meals that can reduce CNI absorption (88). These factors need to be considered when quantifying the AOP. There are limited studies available for each of the KEs and KERs to provide empirical support for this AOP, and there is a lack of substantial information on the dose-response relationships for the stressor agents. Additionally, no single study measures all reported KEs simultaneously following exposure to various doses, which hinders the ability to perform a highly accurate dose-response and concordance analysis. Extensive further research is needed to provide a better quantitative understanding of dose-response relationships between upstream and downstream KEs, as well as the elucidation of the MIE.

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

The quantitave understanding of the KERs does not apply to this qualitative AOP. The Weight of Evidence (WoE) analysis revealed that many KERs lack considerable experimental proof, particularly in demonstrating the direct relationship with the proposed upstream and downstream KEs within individual studies. Therefore, this AOP should be considered as a qualitative contribution and as a backbone to build on in the future until quantitative data becomes available. While some quantitative connections between upstream KEs have been identified in a few studies, the variability in the models used and the dosages chosen makes it difficult to compare studies and draw conclusions. Ideally, future efforts to strengthen WoE should focus on either gathering data from a single system type that shows exposure to a stressor correlating with changes observed in all described KEs or harmonizing the existing evidence in order to identify gaps to be filled and to further investigate.

The AOP described here aims to provide a basic mechanistic framework for developing in vitro assays that could accurately predict quantitatively nephrotoxicity safety assessments. These assays could be part of an integrated testing strategy to minimize the need for repeated dose toxicity studies. Generating quantitative data by measuring all KEs in a single model after exposure to various concentrations of CNI could also accelerate the development of computational predictive methods. With potential overlap between KEs in this AOP with other nephrotoxic agents, the current AOP could offer additional connections for extensive networks to model nephrotoxicity and kidney failure.

Not Specified

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