MIE-A: Direct ligand interaction with nuclear hormone receptors (ERα/ERβ, AR) or interference with steroidogenic enzymes (e.g., CYP19A1) → receptor activation/antagonism or enzyme inhibition.

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

GO:0005739

GO mitochondrion

UBERON:0003133

UBERON reproductive organ

MP:0003674

MP oxidative stress

GO:0006915

GO apoptotic process

GO:0019098

GO reproductive behavior

GO:0032502

GO developmental process

GO:0042698

GO ovulation cycle

WIKI increased

WIKI functional change

WIKI abnormal

Acetaminophen

2016-11-29T18:42:26

Chloroform

2016-11-29T18:42:27

furan

2020-05-01T14:35:22

Platinum

2022-02-04T14:36:54

Aluminum

2022-02-04T14:42:11

Cadmium

2017-10-25T08:33:12

Mercury

2016-11-29T18:42:19

Uranium

2021-08-05T14:28:50

Arsenic

2021-04-27T00:15:21

Silver

2022-02-03T11:20:11

Manganese

2022-02-04T14:47:23

Nickel

2022-02-04T14:47:59

Zinc

2022-02-04T15:05:00

nanoparticles

2016-12-21T09:40:06

WikiUser_26

ApacheUser rodents

9606

NCBI Homo sapiens

WCS_9606

common toxicological species human

10116

NCBI rat

10095

NCBI mice

10090

NCBI Mus musculus

10116

NCBI Rattus norvegicus

6239

NCBI Caenorhabditis elegans

WCS_7955

common ecological species zebrafish

WCS_35525

common ecological species Daphnia magna

8090

NCBI medaka

WCS_90988

common ecological species fathead minnow

WikiUser_15

ApacheUser Sprague-Dawley

MIE-A: Direct ligand interaction with nuclear hormone receptors (ERα/ERβ, AR) or interference with steroidogenic enzymes (e.g., CYP19A1) → receptor activation/antagonism or enzyme inhibition.

Nuclear receptor binding / steroidogenic enzyme interference

Molecular

AOPWiki 2025-11-07T03:44:44

2025-11-07T03:44:44

MIE-B: Photoactivation / photoproduct formation leading to ROS generation

photoproducts generate ROS

Molecular

AOPWiki 2025-11-07T04:00:17

2025-11-07T04:00:17

KE1: Altered steroidogenesis (gene/protein level)

Altered steroidogenesis

Cellular

AOPWiki 2025-11-07T04:04:23

2025-11-07T04:04:23

Increase, Oxidative Stress

Molecular

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

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

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

High Mixed

High

All life stages

High High

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

2026-02-11T07:05:27

Elevated mitochondrial ROS and dysfunction

Mito ROS dysfunction

Cellular

Within the inner mitochondrial membrane, the electron transport chain (ETC) functions to couple the transfer of electrons from NADH and FADH₂ to molecular oxygen with the translocation of protons, ultimately driving ATP synthesis. During this process, partial reduction of oxygen can lead to the generation of ROS, primarily superoxide (O₂•⁻), which can subsequently be converted to other reactive species such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (•OH) (Brand et al. 2010; Brand et al. 2016). Complex I (NADH:ubiquinone oxidoreductase) (Cocheme et al. 2008; Sharma et al. 2009) and complex III (ubiquinol:cytochrome c oxidoreductase) (Castello et al. 2007) are recognized as major sites of ROS production under both physiological and pathophysiological conditions.  <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/10/03/8anqxy4irh_KE1_Fig_1.jpg" /> Fig. 1. Reactive oxygen species. Two molecules of superoxide can react to generate hydrogen peroxide (H2O2) in a reaction known as dismutation, which is accelerated by the enzyme superoxide dismutase (SOD). In the presence of iron, superoxide and H2O2 react to generate hydroxyl radicals. In addition to superoxide, H2O2 and hydroxyl radicals, other reactive oxygen species (ROS) occur in biological systems., which can be generated from singlet oxygen by antibody molecules. The colour coding indicates the reactivity of individual molecules (yellow, limited reactivity; orange, moderate reactivity; red, high reactivity and non-specificity) (Adapted by permission from Macmillan Publishers Ltd, Lambeth, 2004, copyright (2004)).  Complex I and ROS Formation. Complex I is the largest and most intricate component of the ETC, responsible for the transfer of electrons from NADH to ubiquinone (CoQ) (Sharma et al. 2009). This multistep process involves the passage of electrons through a flavin mononucleotide (FMN) and a series of iron–sulfur (Fe–S) clusters before reaching the CoQ-binding site. ROS formation at complex I primarily occurs through two distinct mechanisms: the "forward" and "reverse" electron transport (RET) modes. In the forward mode, electrons flow from NADH through the FMN and Fe–S centers to ubiquinone, which is reduced to ubiquinol. During this process, a small percentage of electrons may leak prematurely to molecular oxygen, generating superoxide, primarily at the FMN site (Cadenas et al. 1977; Turrens et al. 1980; Turrens et al. 1985). This leakage is relatively limited under normal physiological conditions but can increase when the NADH/NAD⁺ ratio is high or when electron flow downstream is inhibited (Wong et al. 2017; Quinlan et al. 2014; Lambert et al. 2004). The RET mechanism is a potent source of ROS, especially under conditions of high proton motive force and elevated levels of reduced CoQ (Miwa et al. 2003). In this scenario, electrons can flow in reverse from ubiquinol back to complex I’s FMN site, leading to substantial ROS generation. This is particularly evident during conditions such as ischemia-reperfusion, when succinate accumulation during ischemia leads to its rapid oxidation and massive ROS production upon reperfusion.  Complex III and ROS Formation. Complex III transfers electrons from ubiquinol to cytochrome c via the Q-cycle, a mechanism involving two distinct ubiquinone-binding sites: the Qo site (outer) and the Qi site (inner). ROS production by complex III is primarily associated with the Qo site, where a semiquinone intermediate can reduce oxygen to superoxide (Dröse et al. 2011; Dröse et al. 2008; Dröse 2009; Votyakova et al. 2001). This occurs on the outer surface of the inner mitochondrial membrane, allowing superoxide to access both the mitochondrial matrix and the intermembrane space.  Under normal conditions, the ROS produced by complex III are relatively low and are efficiently detoxified by mitochondrial antioxidant systems such as superoxide dismutases (SODs) and glutathione peroxidases. However, when electron flow through complex III is perturbed — for instance, by inhibition of the Qo site (e.g., by antimycin A) — the lifetime of the semiquinone intermediate is prolonged, significantly enhancing ROS production.(Quinlan et al. 2011; Sarewicz et al. 2010; Erecinska et al. 1976; Dröse et al. 2008; Zhang et al. 1998; Quinlan et al. 2012).  Physiological vs. Pathophysiological ROS Production. Under physiological conditions, mitochondrial ROS serve as important signaling molecules that modulate processes including hypoxic response, cellular proliferation, and apoptosis (Ullrich et al. 2014; Handy et al. 2012). Low-level ROS production is tightly regulated and balanced by antioxidant defenses, ensuring redox homeostasis. In contrast, pathophysiological conditions such as ischemia-reperfusion injury, neurodegenerative diseases, and metabolic disorders are characterized by dysregulation of ETC components and antioxidant systems (Lin et al. 2000). This leads to excessive and sustained ROS production, causing oxidative damage to mitochondrial DNA, lipids, and proteins, further impairing mitochondrial function and contributing to disease progression (Kowaltoski et al. 2009).  Oxidative Modifications of Mitochondrial Proteins. Mitochondrial proteins, particularly those involved in oxidative phosphorylation (OXPHOS), are highly susceptible to ROS-induced oxPTMs due to their proximity to ROS-generating sites within the electron transport chain (ETC). Cysteine thiol groups are especially reactive and can undergo a range of oxidative modifications, including sulfenylation (–SOH), disulfide bond formation, S-glutathionylation, sulfinylation (–SO₂H), and irreversible sulfonylation (–SO₃H). Additionally, methionine residues may be oxidized to methionine sulfoxide, and tyrosine residues can form dityrosine cross-links or undergo nitration in the presence of peroxynitrite (ONOO⁻). These oxPTMs alter protein conformation, catalytic activity, and protein–protein interactions. For example, oxidation of critical thiols in ATP synthase or adenine nucleotide translocase (ANT) impairs ATP production and disrupts nucleotide exchange across the inner mitochondrial membrane. Similarly, oxidative inactivation of mitochondrial aconitase, a key tricarboxylic acid (TCA) cycle enzyme containing an iron–sulfur cluster, leads to metabolic dysfunction. Proteins of the mitochondrial quality control system, including chaperones and proteases, can also be oxidatively modified, further exacerbating proteotoxic stress and contributing to mitochondrial dysfunction Chung et al. 2013; Mu et al. 2024; Mailloux et al. 2013; Chinta et al. 2011; Brown et al. 2004; Clementi et al. 1998; Nakagawa et al. 2007; Gardner et al. 2002; Tortora et al. 2007; Castro et al. 1994; Aulak et al. 2004; Tretter et al. 1999; Tretter et al. 2000; Yang et al. 2008).  Oxidative Damage to Mitochondrial DNA. Mitochondrial DNA, located in close proximity to the inner mitochondrial membrane where the ETC resides, is highly vulnerable to ROS-induced damage due to its lack of protective histones and limited DNA repair capacity. Oxidative lesions in mtDNA include strand breaks, base modifications such as 8-oxo-2'-deoxyguanosine (8-oxo-dG), and abasic sites. Among these, 8-oxo-dG is a prominent biomarker of oxidative DNA damage and can result in G→T transversions during replication. ROS-induced damage to mtDNA leads to impaired transcription and translation of mitochondrial-encoded subunits of respiratory complexes, which further compromises OXPHOS efficiency and creates a vicious cycle of increasing ROS production. mtDNA mutations and deletions accumulate over time, contributing to aging and degenerative disease phenotypes. Additionally, oxidized mtDNA can be released into the cytosol, where it acts as a damage-associated molecular pattern (DAMP), triggering inflammatory responses via activation of the cGAS–STING pathway or inflammasomes (Cooke et al. 2003; Evans et al. 2004; Floyd et al. 1986; Chen et al. 2010; Sanders et al. 2017; Bender et al. 2006; Sharma et al. 2019; Druzhyna et al. 2008; Hahm et al. 2022; Shokolenko et al. 2014).  Lipid Peroxidation in Mitochondrial Membranes. The mitochondrial inner membrane is rich in polyunsaturated fatty acids, particularly cardiolipin, which is essential for the structural organization and function of respiratory supercomplexes. ROS, especially hydroxyl radicals, initiate lipid peroxidation through the abstraction of hydrogen atoms from lipid acyl chains, generating lipid peroxyl radicals and hydroperoxides. These reactive lipid species propagate oxidative damage across the membrane and compromise its integrity and fluidity. Peroxidation of cardiolipin disrupts the optimal assembly of ETC complexes and impairs cytochrome c anchoring. Oxidized cardiolipin also promotes the release of cytochrome c into the cytosol, a key step in the initiation of intrinsic apoptosis. Moreover, lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) form covalent adducts with mitochondrial proteins, further impairing enzymatic function and contributing to oxidative stress signaling and cell death pathways (Schlame et al. 1993; Chu et al. 2013; Skulachev et al. 2010; Bayir et al. 2006; Milazzo et al. 2017; Xiao et al. 2017; Chen et al. 1994; Mohammadi-Bardbori et al. 2008; Satoh et al. 1997; Chen et al. 2024; Bernardi et al. 2023; Riojas-Hernandez et al. 2015).  Consequences for Mitochondrial Function. Collectively, oxidative modifications of mitochondrial proteins, DNA, and lipids disrupt mitochondrial bioenergetics, including impaired electron transport, ATP depletion, altered redox balance, and dysregulated calcium handling. Mitochondrial membrane potential (Δψm) collapses under severe oxidative stress, leading to the opening of the mitochondrial permeability transition pore (mPTP), mitochondrial swelling, and eventual release of pro-apoptotic factors. These events mark the progression from mitochondrial dysfunction to cell death via apoptosis or necrosis. Furthermore, persistent oxidative damage impairs mitophagy, the selective removal of damaged mitochondria, allowing dysfunctional organelles to accumulate and propagate ROS signaling. In chronic disease states, this contributes to a feed-forward loop of mitochondrial damage, inflammation, and tissue degeneration (Frank et al. 2012).

Amplex Red Ultra assay. The Amplex® Red Ultra assay has emerged as a sensitive and specific technique for the quantitative measurement of hydrogen peroxide (H₂O₂), a primary and relatively stable ROS generated by mitochondria (Grivennikova et al. 2018). The assay is based on the horseradish peroxidase (HRP)-catalyzed reaction between H₂O₂ and the Amplex Red Ultra reagent (10-acetyl-3,7-dihydroxyphenoxazine), producing the highly fluorescent compound resorufin. The fluorescence intensity of resorufin is directly proportional to the amount of H₂O₂ present, allowing real-time monitoring of mitochondrial ROS production. In a mitochondrial context, ROS primarily originate as superoxide anions (O₂•⁻), predominantly produced at complexes I and III of the electron transport chain. These anions are rapidly dismutated to H₂O₂ by mitochondrial superoxide dismutase (SOD2) in the matrix and SOD1 in the intermembrane space. The resultant H₂O₂ diffuses through membranes and accumulates in the surrounding medium, where it can be quantitatively detected using the Amplex Red Ultra assay (Munro et al. 2019; Quinlan et al. 2013). To perform the assay, isolated mitochondria or permeabilized cells are incubated in a reaction buffer containing Amplex Red Ultra, HRP, and appropriate mitochondrial substrates and inhibitors. The choice of substrate (e.g., glutamate/malate for complex I or succinate for complex II) and respiratory conditions (e.g., state 2, 3, or uncoupled respiration) allows the assessment of ROS production under defined bioenergetic states. Inhibitors such as rotenone (complex I), antimycin A (complex III), or myxothiazol can be used to modulate electron flow and enhance ROS formation at specific complexes, enabling mechanistic dissection of ROS sources (Murphy et al. 2022). Importantly, care must be taken to avoid artifacts from exogenous ROS or enzyme autoxidation, and all reactions should be controlled for background fluorescence (Starkov et al. 2010; Tretter et al. 2014). The improved formulation of Amplex Red Ultra offers enhanced resistance to autoxidation and greater photostability compared to conventional Amplex Red, making it particularly suitable for high-sensitivity assays and extended kinetic measurements. The assay is typically performed using a microplate reader set to excitation and emission wavelengths of ~530 nm and ~590 nm, respectively. The high sensitivity of the Amplex Red Ultra assay allows detection of H₂O₂ in the picomolar to low nanomolar range, making it well-suited for studies requiring precise quantification of mitochondrial ROS output.  MitoSOX. MitoSOX™ dyes are a family of fluorogenic probes specifically designed for the selective detection of mitochondrial superoxide in live cells. The most commonly used derivative, MitoSOX™ Red, is a mitochondrial-targeted derivative of hydroethidine (HE), conjugated to a triphenylphosphonium (TPP⁺) moiety that drives its accumulation into the mitochondrial matrix due to the negative membrane potential (Δψm) (Zielonka et al. 2010; Mukhopadhyay et al. 2007). Upon entry into the mitochondria, MitoSOX Red is oxidized specifically by superoxide to form a highly fluorescent ethidium-like product that binds to mitochondrial DNA, emitting red fluorescence upon excitation. The reaction between superoxide and MitoSOX is rapid and relatively selective, although some degree of nonspecific oxidation can occur, particularly in the presence of other oxidants such as hydrogen peroxide, peroxynitrite, or hydroxyl radicals (Roelofs et al. 2015). Therefore, appropriate experimental controls are essential to validate superoxide specificity, including the use of superoxide dismutase mimetics or mitochondrial uncouplers that reduce Δψm and consequently MitoSOX uptake. In practical applications, live cells or isolated mitochondria are incubated with MitoSOX Red under controlled conditions, typically at concentrations of 2–5 μM for 10–30 minutes at 37°C. The cells are then washed to remove excess dye, and fluorescence is detected using fluorescence microscopy, flow cytometry, or plate-based fluorometry. The fluorescence excitation and emission maxima for the oxidized product are approximately 510 nm and 580 nm, respectively (Kauffman et al. 2010). Quantitative analysis requires normalization to mitochondrial content or membrane potential, often using additional stains such as MitoTracker Green or TMRE. MitoSOX Red is particularly valuable in experiments aiming to identify the specific sites or triggers of mitochondrial superoxide production. For instance, mitochondrial ROS generation can be stimulated by respiratory chain inhibitors such as rotenone (complex I) or antimycin A (complex III), redox cyclers which enhance electron leakage and superoxide formation. Similarly, metabolic substrates can modulate ROS production by altering electron flow and redox status within the ETC. In these contexts, MitoSOX allows for real-time detection of dynamic changes in ROS generation in response to pharmacological or genetic perturbations.  Electron Paramagnetic Resonance (EPR). Electron paramagnetic resonance (EPR) spectroscopy, also known as electron spin resonance (ESR), is a powerful and direct analytical technique for the detection of unpaired electrons in paramagnetic species, including free radicals such as reactive oxygen species (ROS). In the context of mitochondrial research, EPR provides a highly specific and quantitative method for detecting and characterizing ROS generated within the mitochondrial respiratory chain, especially under conditions of oxidative stress or during the evaluation of mitochondrial-targeted therapies (Chen et al. 2014). Unlike fluorescence-based probes, which infer ROS presence through secondary reactions, EPR directly detects the presence of short-lived radical species or their stabilized spin-adducts. Native ROS such as superoxide (O₂•⁻), hydroxyl radicals (•OH), and nitric oxide (NO•) are typically too reactive and short-lived to be measured directly by EPR. Therefore, EPR detection of mitochondrial ROS relies primarily on the use of spin-trapping agents (Griendling et al. 2016). These compounds react with transient radicals to form more stable radical adducts that can be detected by EPR. Among the most widely used spin traps for mitochondrial studies is 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which forms stable adducts with O₂•⁻ and •OH. Alternatively, cyclic nitrones such as DEPMPO and EMPO offer enhanced specificity and longer half-lives of the spin-adducts, improving sensitivity in complex biological systems (Hardy et al. 2007; Hardy et al. 2014).   To measure mitochondrial ROS formation using EPR, isolated mitochondria or permeabilized cells are incubated with suitable substrates and respiratory chain inhibitors in the presence of the spin trap. For example, in the presence of succinate and antimycin A, mitochondria generate high levels of ROS at complex III, leading to enhanced spin-adduct formation detectable by EPR. Similarly, reverse electron transport from succinate oxidation can be induced to stimulate superoxide production at complex I, allowing site-specific investigation of ROS sources. EPR spectra provide detailed information on the chemical environment of the radical species, including hyperfine splitting constants and g-values, which help identify the specific type of ROS involved. Quantification is achieved by integrating the signal intensity of the spin-adduct, which is proportional to the radical concentration. To improve signal specificity and reduce background noise, mitochondria-targeted spin traps such as mito-TEMPO or mito-CP (cyclic nitroxides conjugated to lipophilic cations like triphenylphosphonium) can be employed. These agents accumulate within mitochondria due to the membrane potential and provide localized detection of ROS, particularly superoxide. One of the key advantages of EPR is its ability to distinguish between different radical species, a capability that is not matched by conventional fluorescence or chemiluminescence assays. Furthermore, EPR enables real-time kinetic monitoring of radical generation and decay under tightly controlled experimental conditions. However, the technique also presents challenges: it requires specialized instrumentation, careful sample preparation under anaerobic or low-temperature conditions to preserve radical species, and relatively high concentrations of spin traps, which may perturb native redox equilibria.  Coelenterazine chemiluminescence is a sensitive and non-invasive analytical method for detecting elevated mitochondrial reactive oxygen species (ROS), particularly superoxide (O₂•⁻) and hydrogen peroxide (H₂O₂), in intact cells and isolated mitochondria (Lucas et al. 1992; Teranishi et al. 1997). Coelenterazine, a luminescent substrate originally derived from marine organisms such as Renilla and Aequorea, undergoes oxidative decarboxylation upon reaction with ROS to form coelenteramide, emitting photons in the blue spectral range (typically 480–500 nm). The intensity of this light emission is directly proportional to the amount of ROS present, enabling real-time quantification of oxidative stress under physiological and pathophysiological conditions. The utility of coelenterazine for mitochondrial ROS detection lies in its cell-permeable and lipophilic nature, which allows it to diffuse across membranes and accumulate in intracellular compartments, including the mitochondrial matrix. Several coelenterazine derivatives have been developed to enhance specificity and mitochondrial targeting, such as coelenterazine-h and mito-coelenterazine, which exhibit improved sensitivity for mitochondrial superoxide and reduced background luminescence. These derivatives preferentially localize to mitochondria due to their increased hydrophobicity or conjugation with lipophilic cationic moieties, facilitating the detection of ROS specifically originating from the mitochondrial electron transport chain. In experimental applications, cells or mitochondria are incubated with coelenterazine under controlled metabolic conditions. Substrates and inhibitors of the electron transport chain can be used to modulate ROS production at specific sites. For instance, the use of succinate in combination with antimycin A enhances ROS generation at complex III, while reverse electron transport induced by succinate oxidation in the presence of rotenone highlights superoxide production at complex I. The luminescence signal is recorded using highly sensitive luminometers or photon-counting devices, providing kinetic data on ROS production in real time. Coelenterazine chemiluminescence is particularly suited for high-throughput screening and longitudinal studies due to its non-destructive nature and compatibility with live-cell imaging platforms (Tarpey et al. 1999; Daiber et al. 2004). Unlike fluorescence-based ROS probes, which often require excitation light that may introduce phototoxicity or interfere with mitochondrial function, chemiluminescence avoids exogenous illumination and thereby minimizes assay artifacts. Furthermore, the rapid and continuous signal output of coelenterazine enables temporal resolution of ROS dynamics in response to metabolic shifts, pharmacological interventions, or environmental stressors.

Redox cycling is a universal event occurring in any cells of any species as well as in bacteria and yeast (Cocheme and Murphy, 2008). Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (e.g. D. melanogaster and C. elegans) are considered as potential models to study mitochondrial functionality. 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 (Waerzeggers et al., 2010; Winklhofer and Haass, 2010) as well as in humans (Winklhofer and Haass, 2010). brain region-specific mitochondrial membrane potential and susceptibility towards dysfunction of mitochondrial oxidative phosphorylation (OXPHOS) was also observed by Pickrell et al. (2011). Here the striatum was found to be especially sensitive towards disturbance of OXPHOS due to the high striatal mitochondrial OXPHOS and membrane potential, which is prone to collapse when OXPHOS activity is reduced. This instance becomes important when studies on compound effects on isolated mitochondria are not of the correct origin, which would– for studying Parkinsonism– be the brain, and here the nigrostriatal area. In addition to mitochondrial differences between organs and intra-organ regions, species-specific mitochondrial activity was also measured.

UBERON:0000062

UBERON organ

CL:0000255

CL eukaryotic cell

High Male High Female

High

All life stages

High High High

Aulak KS, Koeck T, Crabb JW, Stuehr DJ. Dynamics of protein nitration in cells and mitochondria. Am J Physiol Heart Circ Physiol. 2004 Jan;286(1):H30-8. doi: 10.1152/ajpheart.00743.2003. PMID: 14684358.  Bayir H, Fadeel B, Palladino MJ, Witasp E, Kurnikov IV, Tyurina YY, Tyurin VA, Amoscato AA, Jiang J, Kochanek PM, DeKosky ST, Greenberger JS, Shvedova AA, Kagan VE. Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria. Biochim Biophys Acta. 2006 May-Jun;1757(5-6):648-59. doi: 10.1016/j.bbabio.2006.03.002. Epub 2006 Mar 31. PMID: 16740248. Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, Jaros E, Hersheson JS, Betts J, Klopstock T, Taylor RW, Turnbull DM. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet. 2006 May;38(5):515-7. doi: 10.1038/ng1769. Epub 2006 Apr 9. PMID: 16604074.  Bernardi P, Gerle C, Halestrap AP, Jonas EA, Karch J, Mnatsakanyan N, Pavlov E, Sheu SS, Soukas AA. Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions. Cell Death Differ. 2023 Aug;30(8):1869-1885. doi: 10.1038/s41418-023-01187-0. Epub 2023 Jul 17. PMID: 37460667; PMCID: PMC10406888.  Brand MD. Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling. Free Radic Biol Med. 2016 Nov;100:14-31. doi: 10.1016/j.freeradbiomed.2016.04.001. Epub 2016 Apr 13. PMID: 27085844.  Brand MD. The sites and topology of mitochondrial superoxide production. Exp Gerontol. 2010 Aug;45(7-8):466-72. doi: 10.1016/j.exger.2010.01.003. Epub 2010 Jan 11. PMID: 20064600; PMCID: PMC2879443.  Brown GC, Borutaite V. Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols. Biochim Biophys Acta. 2004 Jul 23;1658(1-2):44-9. doi: 10.1016/j.bbabio.2004.03.016. PMID: 15282173.  Cadenas E, Boveris A, Ragan CI, Stoppani AO. Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch Biochem Biophys. 1977 Apr 30;180(2):248-57. doi: 10.1016/0003-9861(77)90035-2. PMID: 195520.  Castello PR, Drechsel DA, Patel M. Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain. J Biol Chem. 2007 May 11;282(19):14186-93. doi: 10.1074/jbc.M700827200. Epub 2007 Mar 27. PMID: 17389593; PMCID: PMC3088512.  Castro L, Rodriguez M, Radi R. Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J Biol Chem. 1994 Nov 25;269(47):29409-15. PMID: 7961920.  Chen JJ, Yu BP. Alterations in mitochondrial membrane fluidity by lipid peroxidation products. Free Radic Biol Med. 1994 Nov;17(5):411-8. doi: 10.1016/0891-5849(94)90167-8. PMID: 7835747.  Chen Q, Niu Y, Zhang R, Guo H, Gao Y, Li Y, Liu R. The toxic influence of paraquat on hippocampus of mice: involvement of oxidative stress. Neurotoxicology. 2010 Jun;31(3):310-6. doi: 10.1016/j.neuro.2010.02.006. Epub 2010 Mar 6. PMID: 20211647.  Chen S, Li Q, Shi H, Li F, Duan Y, Guo Q. New insights into the role of mitochondrial dynamics in oxidative stress-induced diseases. Biomed Pharmacother. 2024 Sep;178:117084. doi: 10.1016/j.biopha.2024.117084. Epub 2024 Aug 1. PMID: 39088967.  Chinta SJ, Andersen JK. Nitrosylation and nitration of mitochondrial complex I in Parkinson's disease. Free Radic Res. 2011 Jan;45(1):53-8. doi: 10.3109/10715762.2010.509398. Epub 2010 Sep 6. PMID: 20815786.  Chu CT, Ji J, Dagda RK, Jiang JF, Tyurina YY, Kapralov AA, Tyurin VA, Yanamala N, Shrivastava IH, Mohammadyani D, Wang KZQ, Zhu J, Klein-Seetharaman J, Balasubramanian K, Amoscato AA, Borisenko G, Huang Z, Gusdon AM, Cheikhi A, Steer EK, Wang R, Baty C, Watkins S, Bahar I, Bayir H, Kagan VE. Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol. 2013 Oct;15(10):1197-1205. doi: 10.1038/ncb2837. Epub 2013 Sep 15. PMID: 24036476; PMCID: PMC3806088.  Chung HS, Wang SB, Venkatraman V, Murray CI, Van Eyk JE. Cysteine oxidative posttranslational modifications: emerging regulation in the cardiovascular system. Circ Res. 2013 Jan 18;112(2):382-92. doi: 10.1161/CIRCRESAHA.112.268680. PMID: 23329793; PMCID: PMC4340704.  Clementi E, Brown GC, Feelisch M, Moncada S. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7631-6. doi: 10.1073/pnas.95.13.7631. PMID: 9636201; PMCID: PMC22706.  Cochemé HM, Murphy MP. Complex I is the major site of mitochondrial superoxide production by paraquat. J Biol Chem. 2008 Jan 25;283(4):1786-98. doi: 10.1074/jbc.M708597200. Epub 2007 Nov 26. PMID: 18039652.  Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 2003 Jul;17(10):1195-214. doi: 10.1096/fj.02-0752rev. PMID: 12832285.  Dröse S, Bleier L, Brandt U. A common mechanism links differently acting complex II inhibitors to cardioprotection: modulation of mitochondrial reactive oxygen species production. Mol Pharmacol. 2011 May;79(5):814-22. doi: 10.1124/mol.110.070342. Epub 2011 Jan 28. PMID: 21278232.  Dröse S, Brandt U. The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex. J Biol Chem. 2008 Aug 1;283(31):21649-54. doi: 10.1074/jbc.M803236200. Epub 2008 Jun 3. PMID: 18522938.  Dröse S, Hanley PJ, Brandt U. Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III. Biochim Biophys Acta. 2009 Jun;1790(6):558-65. doi: 10.1016/j.bbagen.2009.01.011. Epub 2009 Feb 6. PMID: 19364480.  Druzhyna NM, Wilson GL, LeDoux SP. Mitochondrial DNA repair in aging and disease. Mech Ageing Dev. 2008 Jul-Aug;129(7-8):383-90. doi: 10.1016/j.mad.2008.03.002. Epub 2008 Mar 13. PMID: 18417187; PMCID: PMC2666190.  Erecińska M, Wilson DF. The effect of antimycin A on cytochromes b561, b566, and their relationship to ubiquinone and the iron-sulfer centers S-1 (+N-2) and S-3. Arch Biochem Biophys. 1976 May;174(1):143-57. doi: 10.1016/0003-9861(76)90333-7. PMID: 180891.  Evans MD, Dizdaroglu M, Cooke MS. Oxidative DNA damage and disease: induction, repair and significance. Mutat Res. 2004 Sep;567(1):1-61. doi: 10.1016/j.mrrev.2003.11.001. PMID: 15341901. Farooqui T, Farooqui AA, 2012. Oxidative stress in Vertebrates and Invertebrate: Molecular Aspects of Cell Signalling. Wiley-Blackwell, Chapter 27, pp. 377–385. Floyd RA, Watson JJ, Wong PK, Altmiller DH, Rickard RC. Hydroxyl free radical adduct of deoxyguanosine: sensitive detection and mechanisms of formation. Free Radic Res Commun. 1986;1(3):163-72. doi: 10.3109/10715768609083148. PMID: 2577733.   Frank M, Duvezin-Caubet S, Koob S, Occhipinti A, Jagasia R, Petcherski A, Ruonala MO, Priault M, Salin B, Reichert AS. Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner. Biochim Biophys Acta. 2012 Dec;1823(12):2297-310. doi: 10.1016/j.bbamcr.2012.08.007. Epub 2012 Aug 16. PMID: 22917578.  Gardner PR. Aconitase: sensitive target and measure of superoxide. Methods Enzymol. 2002;349:9-23. doi: 10.1016/s0076-6879(02)49317-2. PMID: 11912933.  Hahm JY, Park J, Jang ES, Chi SW. 8-Oxoguanine: from oxidative damage to epigenetic and epitranscriptional modification. Exp Mol Med. 2022 Oct;54(10):1626-1642. doi: 10.1038/s12276-022-00822-z. Epub 2022 Oct 21. PMID: 36266447; PMCID: PMC9636213.  Handy DE, Loscalzo J. Redox regulation of mitochondrial function. Antioxid Redox Signal. 2012 Jun 1;16(11):1323-67. doi: 10.1089/ars.2011.4123. Epub 2012 Feb 3. PMID: 22146081; PMCID: PMC3324814.  Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radic Biol Med. 2009 Aug 15;47(4):333-43. doi: 10.1016/j.freeradbiomed.2009.05.004. Epub 2009 May 8. PMID: 19427899.  Lambert AJ, Brand MD. Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I). J Biol Chem. 2004 Sep 17;279(38):39414-20. doi: 10.1074/jbc.M406576200. Epub 2004 Jul 15. PMID: 15262965.  Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006 Oct 19;443(7113):787-95. doi: 10.1038/nature05292. PMID: 17051205.  Mailloux RJ, Jin X, Willmore WG. Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions. Redox Biol. 2013 Dec 19;2:123-39. doi: 10.1016/j.redox.2013.12.011. PMID: 24455476; PMCID: PMC3895620.  Martinez-Cruz O, Sanchez-Paz A, Garcia-Carre~ no F, Jimenez-Gutierrez L, Navarrete del Toro Mde L and Muhlia-Almazan A, 2012. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com). In: Clark K (ed.). Bioenergetics, ISBN 978-953-51-0090-4, InTech, pp. 181–218.  Milazzo L, Tognaccini L, Howes BD, Sinibaldi F, Piro MC, Fittipaldi M, Baratto MC, Pogni R, Santucci R, Smulevich G. Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only. Biochemistry. 2017 Apr 4;56(13):1887-1898. doi: 10.1021/acs.biochem.6b01281. Epub 2017 Mar 20. PMID: 28277678.  Miwa S, St-Pierre J, Partridge L, Brand MD. Superoxide and hydrogen peroxide production by Drosophila mitochondria. Free Radic Biol Med. 2003 Oct 15;35(8):938-48. doi: 10.1016/s0891-5849(03)00464-7. PMID: 14556858.  Mohammadi-Bardbori A, Ghazi-Khansari M. Alternative electron acceptors: Proposed mechanism of paraquat mitochondrial toxicity. Environ Toxicol Pharmacol. 2008 Jul;26(1):1-5. doi: 10.1016/j.etap.2008.02.009. Epub 2008 Feb 29. PMID: 21783880.  Mu B, Zeng Y, Luo L, Wang K. Oxidative stress-mediated protein sulfenylation in human diseases: Past, present, and future. Redox Biol. 2024 Oct;76:103332. doi: 10.1016/j.redox.2024.103332. Epub 2024 Aug 30. PMID: 39217848; PMCID: PMC11402764.  Nakagawa H, Komai N, Takusagawa M, Miura Y, Toda T, Miyata N, Ozawa T, Ikota N. Nitration of specific tyrosine residues of cytochrome C is associated with caspase-cascade inactivation. Biol Pharm Bull. 2007 Jan;30(1):15-20. doi: 10.1248/bpb.30.15. PMID: 17202652.  Pickrell AM, Fukui H, Wang X, Pinto M, Moraes CT, 2011. The striatum is highly susceptible to mitochondrial oxidative phosphorylation dysfunctions. Journal of Neuroscience, 31, 9895–9904.  Quinlan CL, Gerencser AA, Treberg JR, Brand MD. The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle. J Biol Chem. 2011 Sep 9;286(36):31361-72. doi: 10.1074/jbc.M111.267898. Epub 2011 Jun 27. PMID: 21708945; PMCID: PMC3173136.  Quinlan CL, Goncalves RL, Hey-Mogensen M, Yadava N, Bunik VI, Brand MD. The 2-oxoacid dehydrogenase complexes in mitochondria can produce superoxide/hydrogen peroxide at much higher rates than complex I. J Biol Chem. 2014 Mar 21;289(12):8312-25. doi: 10.1074/jbc.M113.545301. Epub 2014 Feb 10. PMID: 24515115; PMCID: PMC3961658.  Quinlan CL, Treberg JR, Perevoshchikova IV, Orr AL, Brand MD. Native rates of superoxide production from multiple sites in isolated mitochondria measured using endogenous reporters. Free Radic Biol Med. 2012 Nov 1;53(9):1807-17. doi: 10.1016/j.freeradbiomed.2012.08.015. Epub 2012 Aug 17. PMID: 22940066; PMCID: PMC3472107.  Riojas-Hernández A, Bernal-Ramírez J, Rodríguez-Mier D, Morales-Marroquín FE, Domínguez-Barragán EM, Borja-Villa C, Rivera-Álvarez I, García-Rivas G, Altamirano J, García N. Enhanced oxidative stress sensitizes the mitochondrial permeability transition pore to opening in heart from Zucker Fa/fa rats with type 2 diabetes. Life Sci. 2015 Nov 15;141:32-43. doi: 10.1016/j.lfs.2015.09.018. Epub 2015 Sep 25. PMID: 26407476.  Sanders LH, Paul KC, Howlett EH, Lawal H, Boppana S, Bronstein JM, Ritz B, Greenamyre JT. Editor's Highlight: Base Excision Repair Variants and Pesticide Exposure Increase Parkinson's Disease Risk. Toxicol Sci. 2017 Jul 1;158(1):188-198. doi: 10.1093/toxsci/kfx086. PMID: 28460087; PMCID: PMC6075191.  Sarewicz M, Borek A, Cieluch E, Swierczek M, Osyczka A. Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc₁. Implications for the mechanism of superoxide production. Biochim Biophys Acta. 2010 Nov;1797(11):1820-7. doi: 10.1016/j.bbabio.2010.07.005. Epub 2010 Jul 15. PMID: 20637719; PMCID: PMC3057645.  Satoh T, Enokido Y, Aoshima H, Uchiyama Y, Hatanaka H. Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells. J Neurosci Res. 1997 Nov 1;50(3):413-20. doi: 10.1002/(SICI)1097-4547(19971101)50:3<413::AID-JNR7>3.0.CO;2-L. PMID: 9364326.  Schlame M, Haldar D. Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria. J Biol Chem. 1993 Jan 5;268(1):74-9. PMID: 8380172.  Sharma P, Sampath H. Mitochondrial DNA Integrity: Role in Health and Disease. Cells. 2019 Jan 29;8(2):100. doi: 10.3390/cells8020100. PMID: 30700008; PMCID: PMC6406942.  Sharma LK, Lu J, Bai Y. Mitochondrial respiratory complex I: structure, function and implication in human diseases. Curr Med Chem. 2009;16(10):1266-77. doi: 10.2174/092986709787846578. PMID: 19355884; PMCID: PMC4706149.  Shokolenko IN, Wilson GL, Alexeyev MF. Aging: A mitochondrial DNA perspective, critical analysis and an update. World J Exp Med. 2014 Nov 20;4(4):46-57. doi: 10.5493/wjem.v4.i4.46. PMID: 25414817; PMCID: PMC4237642.  Skulachev VP, Antonenko YN, Cherepanov DA, Chernyak BV, Izyumov DS, Khailova LS, Klishin SS, Korshunova GA, Lyamzaev KG, Pletjushkina OY, Roginsky VA, Rokitskaya TI, Severin FF, Severina II, Simonyan RA, Skulachev MV, Sumbatyan NV, Sukhanova EI, Tashlitsky VN, Trendeleva TA, Vyssokikh MY, Zvyagilskaya RA. Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs). Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):878-89. doi: 10.1016/j.bbabio.2010.03.015. Epub 2010 Mar 20. PMID: 20307489.  Tórtora V, Quijano C, Freeman B, Radi R, Castro L. Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation. Free Radic Biol Med. 2007 Apr 1;42(7):1075-88. doi: 10.1016/j.freeradbiomed.2007.01.007. Epub 2007 Jan 8. PMID: 17349934.  Tretter L, Adam-Vizi V. Inhibition of alpha-ketoglutarate dehydrogenase due to H2O2-induced oxidative stress in nerve terminals. Ann N Y Acad Sci. 1999;893:412-6. doi: 10.1111/j.1749-6632.1999.tb07867.x. PMID: 10672279 Tretter L, Adam-Vizi V. Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress. J Neurosci. 2000 Dec 15;20(24):8972-9. doi: 10.1523/JNEUROSCI.20-24-08972.2000. PMID: 11124972; PMCID: PMC6773008.  Turrens JF, Alexandre A, Lehninger AL. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys. 1985 Mar;237(2):408-14. doi: 10.1016/0003-9861(85)90293-0. PMID: 2983613.  Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980 Nov 1;191(2):421-7. doi: 10.1042/bj1910421. PMID: 6263247; PMCID: PMC1162232.  Ullrich V, Schildknecht S. Sensing hypoxia by mitochondria: a unifying hypothesis involving S-nitrosation. Antioxid Redox Signal. 2014 Jan 10;20(2):325-38. doi: 10.1089/ars.2012.4788. Epub 2012 Sep 11. PMID: 22793377.  Votyakova TV, Reynolds IJ. DeltaPsi(m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria. J Neurochem. 2001 Oct;79(2):266-77. doi: 10.1046/j.1471-4159.2001.00548.x. PMID: 11677254.  Waerzeggers, Yannic Monfared, Parisa Viel, Thomas Winkeler, Alexandra Jacobs, Andreas H, 2010. Mouse models in neurological disorders: applications of non-invasive imaging. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease, 1802, 819–839.  Winklhofer K, Haass C, 2010. Mitochondrial dysfunction in Parkinson’s disease. Biochimica et Biophysica Acta (BBA)- Molecular Basis of Disease, 1802, 29–44.  Wong HS, Dighe PA, Mezera V, Monternier PA, Brand MD. Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions. J Biol Chem. 2017 Oct 13;292(41):16804-16809. doi: 10.1074/jbc.R117.789271. Epub 2017 Aug 24. PMID: 28842493; PMCID: PMC5641882.  Xiao M, Zhong H, Xia L, Tao Y, Yin H. Pathophysiology of mitochondrial lipid oxidation: Role of 4-hydroxynonenal (4-HNE) and other bioactive lipids in mitochondria. Free Radic Biol Med. 2017 Oct;111:316-327. doi: 10.1016/j.freeradbiomed.2017.04.363. Epub 2017 Apr 27. PMID: 28456642.  Yang ES, Lee JH, Park JW. Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase. Biochimie. 2008 Sep;90(9):1316-24. doi: 10.1016/j.biochi.2008.03.001. Epub 2008 Mar 19. PMID: 18405671.  Zhang L, Yu L, Yu CA. Generation of superoxide anion by succinate-cytochrome c reductase from bovine heart mitochondria. J Biol Chem. 1998 Dec 18;273(51):33972-6. doi: 10.1074/jbc.273.51.33972. PMID: 9852050.  AOPWiki 2025-09-05T04:58:55

2025-10-03T11:24:55

Apoptosis

Cellular

Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “physiological cell death”, is involved in cell turnover, physiological involution, and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1-/- ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. An AOP focuses existes on p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017]. Apoptosis is defined as a programmed cell death.  A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe).  Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell.    In mammals, the foetal ovary produces hundreds of thousands of oocytes. But most of them die before birth due to apoptosis (Kaur, S., & Kurokawa, M., 2023). The apoptotic process has a specific pattern at different stages: in foetal ovaries, the majority of apoptotic activity was found in germ cells, whereas in adult quiescent cortical follicles, apoptosis occurred from both granulosa and oocyte cells. The oocyte has been shown to be the one that triggers the apoptotic process and causes follicular atresia (Jin, X., et al. (2011). In humans, the primordial follicles' ovarian endowment is formed throughout foetal development. Apoptotic cell death, which is carried out with the assistance of multiple players and routes conserved from worms to humans, depletes this endowment by at least two-thirds prior to birth. As of right now, apoptosis has been linked to atresia, oocyte loss/selection, folliculogenesis, and oogenesis (Hussein MR, 2005) The Bcl-2 is a protein family suppressing apoptosis by binding and inhibiting two proapoptotic proteins (Bax and Bak) and transferring them to the mitochondrial outer membrane. In the absence of inhibition by Bcl2, Bax and Bak destroy the mitochondrial membrane and releases proapoptotic signaling proteins, such as cytochrome c which activated the caspase system. An increased expression of these antiapoptotic proteins (Bcl-2, Bcl-xL) occurs in cancer (Hanahan, Adams, Lowe). Several others pathways such as the loss of TP53 tumor suppressor function, or the increase of survival signals (Igf1/2), or decrease of proapoptotic factors (Bax, Bim, Puma) can also increase tumor growth (Hanahan, Juntilla). In breast cancer a decrease in apoptosis and a resistance to cell death has been described thoroughly, especially using a dysregulation of the Bcl2 system or TP53 (Parton, Williams, Shahbandi).

Apoptosis is characterized by many morphological and biochemical changes such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003]. ・DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003]. ・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli et al., 2014]. ・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli et al., 2014]. ・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli et al., 2014]. ・Cleavage of PARP is detected with Western blotting [Parajuli et al., 2014]. ・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu et al., 2016]. ・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016]. ・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994]. ・For the detection of apoptosis, the testes are fixed in neutral buffered formalin and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008]

・Apoptosis is induced in human prostate cancer cell lines (Homo sapiens) [Parajuli et al., 2014]. ・Apoptosis occurs in B6C3F1 mouse (Mus musculus) [Elmore, 2007]. ・Apoptosis occurs in Sprague-Dawley rat (Rattus norvegicus) [Elmore, 2007]. ・Apoptosis occurs in the nematode (Caenorhabditis elegans) [Elmore, 2007]. <ul> <li>Apoptosis occurs in breast cancer cells, human and mouse (Parton)</li> <li style="text-align:justify">Apoptosis applicable to fishes, hence be used to study as models (dos Santos, N. M., et al. (2008).</li> <li style="text-align:justify">Apoptosis in humans and baboon ovaries (Kugu, K., et al. (1998)</li> <li style="text-align:justify">Apoptosis in amphibians during metamorphosis (Ishizuya-Oka, A., et al. (2010).</li> <li style="text-align:justify">Apoptosis in Drosophila melanogaster (Steller, H. (2008)</li> <li style="text-align:justify">Apoptosis is a highly conserved and essential process across a broad taxonomic range, from unicellular eukaryotes to complex multicellular animals, it is also evident in metazoans (Suraweera, C. D., et al. (2022).</li> <li> Sex Applicability: Both sexes. Apoptosis occurs in male and female systems (e.g., oocyte and sperm cell turnover). </li> <li> Life Stage Applicability: All stages. Especially critical during embryonic development and in maintaining adult tissue homeostasis. </li> </ul>

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific

High

Not Otherwise Specified

High High High High

Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283 Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516 Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163 Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257 Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556 Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004 Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313 Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299 Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43 Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831 Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143 Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052 Yasuhara, S. et al. (2003), "Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis", J Histochem Cytochem 51:873-885 Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181   Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230 Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981. Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline">https://doi.org/10.1038/nature03098</a> Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573. Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747. Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133. Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230 Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981. Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline">https://doi.org/10.1038/nature03098</a> Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573. Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747. Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133. Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.   Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.   <ul> <li>Kaur S, Kurokawa M. Regulation of Oocyte Apoptosis: A View from Gene Knockout Mice. Int J Mol Sci. 2023;24(2).</li> <li>Jin X, Xiao LJ, Zhang XS, Liu YX. Apotosis in ovary. Front Biosci (Schol Ed). 2011;3(2):680-97.</li> <li>Hussein MR. Apoptosis in the ovary: molecular mechanisms. Hum Reprod Update. 2005;11(2):162-77.</li> <li>dos Santos NM, do Vale A, Reis MI, Silva MT. Fish and apoptosis: molecules and pathways. Curr Pharm Des. 2008;14(2):148-69.</li> <li>Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao XJ, Martimbeau S, et al. Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ. 1998;5(1):67-76.</li> <li>Ishizuya-Oka A, Hasebe T, Shi YB. Apoptosis in amphibian organs during metamorphosis. Apoptosis. 2010;15(3):350-64.</li> <li>Steller H. Regulation of apoptosis in Drosophila. Cell Death & Differentiation. 2008;15(7):1132-8.</li> <li>Suraweera CD, Banjara S, Hinds MG, Kvansakul M. Metazoans and Intrinsic Apoptosis: An Evolutionary Analysis of the Bcl-2 Family. International Journal of Molecular Sciences. 2022;23(7):3691.</li> </ul> AOPWiki 2017-02-07T13:21:50

2025-05-31T08:50:09

KE5: Altered circulating reproductive hormones

reproductive hormonal Imbalance

Organ

AOPWiki 2025-11-07T04:17:05

2025-11-07T04:17:05

Reduced fertility / fecundity

Reduced fertility

Individual

AOPWiki 2025-11-07T04:20:22

2025-11-07T04:20:22

Altered, Reproductive behaviour

Individual

AOPWiki 2016-11-29T18:41:24

2016-12-03T16:33:26

Impaired development of, Reproductive organs

Individual

AOPWiki 2016-11-29T18:41:24

2016-12-03T16:33:26

Reduce, Sperm count

Individual

AOPWiki 2020-04-06T09:49:03

2020-04-06T11:40:28

Ovulation of oocytes Reduced, Delayed, or Blocked, Decrease or delay in LH surge required for ovulation

Reduced Ovulation of oocytes

Individual

AOPWiki 2016-11-29T18:41:28

2020-05-19T16:39:16

<upstream-id>b1d0b080-b3e2-4ed6-8981-47d922da0b48</upstream-id> <downstream-id>dcdbafbf-24b9-49ae-83ae-1fb4f1807e3b</downstream-id>

AOPWiki 2025-11-07T04:29:22

2025-11-07T04:29:22

<upstream-id>76071ee6-973f-4e40-962e-eea191e5d362</upstream-id> <downstream-id>dcdbafbf-24b9-49ae-83ae-1fb4f1807e3b</downstream-id>

AOPWiki 2025-11-07T04:43:12

2025-11-07T04:43:12

<upstream-id>76071ee6-973f-4e40-962e-eea191e5d362</upstream-id> <downstream-id>78139536-647c-4cb8-a401-528bee5b8573</downstream-id>

AOPWiki 2025-11-07T04:35:08

2025-11-07T04:35:08

<upstream-id>76071ee6-973f-4e40-962e-eea191e5d362</upstream-id> <downstream-id>72e7790f-8ee4-4b6d-a706-ac4f46a0f6cd</downstream-id>

AOPWiki 2025-11-07T04:43:28

2025-11-07T04:43:28

<upstream-id>78139536-647c-4cb8-a401-528bee5b8573</upstream-id> <downstream-id>72e7790f-8ee4-4b6d-a706-ac4f46a0f6cd</downstream-id>

AOPWiki 2025-11-07T04:36:14

2025-11-07T04:36:14

<upstream-id>72e7790f-8ee4-4b6d-a706-ac4f46a0f6cd</upstream-id> <downstream-id>dbbd7f8a-a03a-4646-95a1-4bac18db17c6</downstream-id>

AOPWiki 2025-11-07T04:37:07

2025-11-07T04:37:07

<upstream-id>dcdbafbf-24b9-49ae-83ae-1fb4f1807e3b</upstream-id> <downstream-id>d8f56292-5ac7-4bd2-9455-4562619ee078</downstream-id>

AOPWiki 2025-11-07T04:37:53

2025-11-07T04:37:53

<upstream-id>dbbd7f8a-a03a-4646-95a1-4bac18db17c6</upstream-id> <downstream-id>2e061416-283f-452f-a1e6-62a965224743</downstream-id>

AOPWiki 2025-11-07T04:38:32

2025-11-07T04:38:32

<upstream-id>d8f56292-5ac7-4bd2-9455-4562619ee078</upstream-id> <downstream-id>2e061416-283f-452f-a1e6-62a965224743</downstream-id>

AOPWiki 2025-11-07T04:39:17

2025-11-07T04:39:17

organic UV filter and its Photoproducts reproductive toxicity pathways

OUVFs and PTP AOP pathways in reproductive toxicity

Brendan Ferreri-Hanberry

2.7

adjacent

Moderate

High adjacent

Low

Moderate adjacent

Moderate

Moderate adjacent

Low

Moderate adjacent

Moderate

High adjacent

High

High adjacent

High

High non-adjacent

Not Specified

Not Specified non-adjacent

Not Specified

Low

High

Embryo

High

Development

High High High High High High

AOPWiki 2025-11-06T04:55:02

2026-06-26T16:12:16