Succinate dehydrogenase, inhibited

117-81-7

BJQHLKABXJIVAM-UHFFFAOYNA-N

BJQHLKABXJIVAM-UHFFFAOYSA-N

Di(2-ethylhexyl) phthalate

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

DTXSID5020607

51-52-5

KNAHARQHSZJURB-UHFFFAOYSA-N

6-Propyl-2-thiouracil

6-Propyl-2 thiouracil (PTU) 4(1H)-Pyrimidinone, 2,3-dihydro-6-propyl-2-thioxo- 2,3-Dihydro-6-propyl-2-thioxo-4(1H)-pyrimidinone 2-Mercapto-4-hydroxy-6-n-propylpyrimidine 2-Mercapto-4-hydroxy-6-propylpyrimidine 2-Mercapto-6-propylpyrimidin-4-ol 2-Thio-4-oxo-6-propyl-1,3-pyrimidine 2-Thio-6-propyl-1,3-pyrimidin-4-one 6-n-Propyl-2-thiouracil 6-n-Propylthiouracil 6-Propyl-2-thio-2,4(1H,3H)pyrimidinedione 6-Propylthiouracil NSC 6498 NSC 70461 Procasil Propacil propiltiouracilo Propycil Propyl-Thiorist Propylthiorit propylthiouracil Propylthiouracile Propyl-Thyracil Prothiucil Prothiurone Prothycil Prothyran Protiural Thiuragyl Thyreostat II URACIL, 4-PROPYL-2-THIO- Uracil, 6-propyl-2-thio-

DTXSID5021209

60-56-0

PMRYVIKBURPHAH-UHFFFAOYSA-N

Methimazole

2H-Imidazole-2-thione, 1,3-dihydro-1-methyl- 1,3-Dihydro-1-methyl-2H-imidazole-2-thione 1-Methyl-1,3-dihydroimidazole-2-thione 1-Methyl-1H-imidazole-2-thiol 1-Methyl-2-mercapto-1H-imidazole 1-Methyl-2-mercaptoimidazole 1-Methyl-4-imidazoline-2-thione 1-Methylimidazole-2(3H)-thione 1-Methylimidazole-2-thiol 1-Methylimidazole-2-thione 2-Mercapto-1-methyl-1H-imidazole 2-Mercapto-1-methylimidazole 2-Mercapto-N-methylimidazole 4-Imidazoline-2-thione, 1-methyl- Basolan Danantizol Favistan Frentirox Imidazole-2-thiol, 1-methyl- Mercaptazole Mercazole Mercazolyl Metazolo Methimazol Methylmercaptoimidazole Metothyrin Metothyrine Metotirin N-Methyl-2-mercaptoimidazole N-Methylimidazolethiol NSC 38608 Strumazol Tapazole Thacapzol Thiamazol thiamazole Thycapzol Thymidazol Thymidazole tiamazol

DTXSID4020820

14797-73-0

VLTRZXGMWDSKGL-UHFFFAOYSA-M

Perchlorate

Perchlorate ion Perchlorate ion (ClO41-) Perchlorate ion(1-) Perchlorate(1-) Perchloric acid, ion(1-)

DTXSID6024252

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:0045281

GO succinate dehydrogenase complex

CHEBI:30660

CHEBI thyroxine

CHEBI:18421

CHEBI superoxide

CHEBI:60311

CHEBI thyroid hormone

GO:0000104

GO succinate dehydrogenase activity

GO:0046443

GO FAD metabolic process

GO:0006105

GO succinate metabolic process

HP:0000855

HP Insulin resistance

MP:0005475

MP abnormal circulating thyroxine level

GO:0022904

GO respiratory electron transport chain

GO:0006590

GO thyroid hormone generation

WIKI decreased

WIKI increased

Di(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Mono(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Propylthiouracil

2016-11-29T18:42:22

Methimazole

2016-11-29T18:42:19

Perchlorate

2016-11-29T18:42:26

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

10116

NCBI rat

WCS_9606

common toxicological species human

10090

NCBI mouse

WCS_9031

common ecological species chicken

WCS_8355

common ecological species Xenopus laevis

WCS_7955

common ecological species zebrafish

WCS_90988

common ecological species fathead minnow

9823

NCBI Sus scrofa

WikiUser_17

mammals

Succinate dehydrogenase, inhibited

SDH, inhibited

Molecular

Eukaryotic succinate dehydrogenase (SDH, EC1.3.5.1 (Brenda, IntEnz)) is an enzyme complex comprising four polypeptide chains (SDHA - SDHD) with associated FAD,  Fe-S and haem prosthetic groups that catalyses the reversible oxidation (dehydrogenation) of succinate to fumarate with concomitant reduction of ubiquinone to ubiquinol, serving to channel reducing equivalents from succinate, a tricarboxylic acid (TCA) cycle intermediate, to ubiquinol, an intermediate of the mitochondrial electron transfer chain (Du et al, 2023). The overall reaction: succinate + ubiquinone = fumarate + ubiquinol comprises two, reversible half-reactions: (1) succinate + FAD = fumarate + FADH2 and: (2) FADH2 + ubiquinone = FAD + ubiquinol each of which is catalysed at a different active site. The active site of reaction 1 is in the hydrophilic protein SDHA that contains the covalently bound FAD group, and protudes from the inner mitochondrial membrane (IMM) into the mitochondrial matrix, making it available to exchange succinate and fumarate within the TCA cycle. The active site of reaction 2 is in a more hydrophobic region comprising transmembrane domains of proteins SDHC and SCHD that insert complex II into the IMM (Du et al, 2023), making it available to ubiquinol and ubiquinone shuttling within the IMM. The presence of two distinct and different active sites enables SDH inibition to be effected in at least two ways: by inhibition of either active site, with potentially different biochemical and physiological consequences, and by inhibitors with differing characteristics. Inhibition of SDH can result in reduction of mitochondrial electron transport, and subsequent inhibition of oxidative phosphorylation (e.g. Chen et al, 2021), and also generation of superoxide in the mitochondria, leading to with subsequently deleterious effects such as initiation of apoptosis or necrosis (Murphy et al, 2009).

Succinate dehydrogenase activity is generally measured by the spectrophotometric detection of colour change in the presence of an  electron acceptor, with succinate (succinic acid) as substrate. Alteration in rate of colour change in the presence of a putative inhibitor determining the strength of that inhibition. The fact that the overall reaction is comprised of two consecutive sub-reactions enables the rate of each sub-reaction - and their inhibition - to be measured separately by appropriate choice of electron acceptor in the presence of succinate as a substrate (e.g. Miyadera et al, 2003). Activities are frequently measured in isolated mitochondria, in order to reduce interference by extra-cytosolic enaymes and intermediates; mitochondria can be sonicated to obviate rate limitation by mitochondrial upake of succinate (e.g. Guo et al, 2016). SDH activity Succinate dehydrogenase (SDH) activity corresponds to reaction (1), above. It can be measured by use of the water-soluble dye 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) in the presence of the intermediate electron carrier phenazine methosulfate (PMS), to intercept electrons before their transport to ubiquinone, and convey them to MTT, which changes colour following its reduction.   SQR activity Succinate quinone reductase (SQR) activity corresponds to the overall reaction (i.e. 1 and 2), above. It can be measured by reduction of 2,6-dichlorophenolindophenol (DCPIP) in the presence of the 2,3-dimethoxy-6-methyl-1,4-benzoquinone (UQ2), which accepts electrons from the ubiquinone reduction site and transfers them to DCPIP, thus being a measure of the rate of the entire reaction catalysed by complex II.

SDH inhibition by phthalate esters has been measured and quantified in mitochondria of hepatocytes of adult male CD rats (Melnick and Schiller, 1982; Melnick and Schiller, 1985). Inter-species differences in SDH structure may lead to different susceptibilities in different taxa. SDH inhibition has been demonstrated by lonidamine, 3-nitroproprionic acid (3-NPA) and 2-thenoyltrifluoroacetone (TTFA) in DB-1, HepG2, HCT116 and HeLa cells, and by lonidamine in mitochondria isolated from adult mouse liver (Guo et al, 2016).

UBERON:0001280

UBERON liver parenchyma

CL:0000182

CL hepatocyte

High Male

High

Adult

High

Brenda, "Information on EC 1.3.5.1 - succinate dehydrogenase", <a href="https://www.brenda-enzymes.org/enzyme.php?ecno=1.3.5.1">https://www.brenda-enzymes.org/enzyme.php?ecno=1.3.5.1</a>, accessed 28/04/2023. Chen, L. et al (2021) "Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation", Journal of Biological Chemistry, Vol 296, 100515 Du, Z. et al (2023) "Structure of the human respiratory complex II", Proceedings of the National Academy of Sciences", Vol 120, e2216713120. Guo, L. et al (2016) "Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine", Journal of Biological Chemistry, Vol 291. pp42-57. IntEnz, "IntEnz Enzyme Nomenclature, EC 1.3.5.1", <a href="https://www.ebi.ac.uk/intenz/query?cmd=SearchID&id=1525&view=INTENZ">https://www.ebi.ac.uk/intenz/query?cmd=SearchID&id=1525&view=INTENZ</a>, accessed 28/04/2023. Melnick, R.L. and Schiller, C.M. (1982), "Mitochondrial toxicity of phthalate esters", Environmental Healh Perspectives, Vol 45, pp51-56. Melnick, R.L. and Schiller, C.M. (1985), "Effect of phthalate esters on energy coupling and succinate oxidation in rat liver mitochondria", Toxicology, Vol 34, pp13-27. Miyadera, H. et al (2003) "Atpenins, potent and specific inhibitors of mitochondrial complex II (succinateubiquinone oxidoreductase)", Proceedings of the National Academy of Sciences, Vol 100, pp473-477. Murphy, M.P. (2009), "How mitochondria produce reactive oxygen species", Biochemical Journal, Vol 417, pp1-13. AOPWiki 2023-04-03T10:56:31

2023-05-04T11:49:24

Insulin resistance, increased

Individual

Under normal physiological conditions, an increase in plasma glucose, for instance in the post-prandial phase, leads to increased insulin secretion by the pancreas, and consequent increase in plasma insulin concentration. The insulin increases glucose uptake into peripheral tissues, and inhibits hepatic glycogenolysis and gluconeogenesis. In times of stress, the insulin response can be inhibited by stress hormones and proinhibitory cytokines, a mechanism known as insulin resistance (IR), that serves to maintain blood glucose levels during stress response (Tsatsoulis et al, 2013). Inappropriate stress hormone and cytokine generation can lead to individuals showing inappropriate insulin resistance, manifested as lack of insulin-stimulation of glucose uptake into adipose and muscle, and lack of insulin suppression of hepatic glucose release.  IR is associated with multiple health conditions including obesity, type 2 diabetes mellitus, metabolic syndrome, cardiovascular disease, metabolism associated fatty liver disease and polycystic ovary syndrome. It is a complex metabolic disorder, the exact causes of which are still to be fully resolved (Li et al, 2022). The mechanism by which IR manifests itself is primarily by abnormalities in insulin signal transduction (Pei et al, 2022).

Clinical detection of insulin resistance generally requires measurement of plasma insulin and/or glucose under controlled circumstances, or alongside specific interventions designed to elicit a physiological insulin response. Multiple methods of differing complexity, technical challenge, invasiveness and reliability have been devised (see Sharma et al, 2020 for a review). The hyperinsulineamic euglycaemic clamp (HEC), involving intravenous infusion of insulin to reach steady state glycemic is considered the gold standard, but is challenging, expensive and invasive. Other methods, such as the homeostatic model for insulin resistance (HOMA-IR), a single sample method, are simpler and less invasive, but show greater interindividual variability and only moderate agreement with the HEC. They are more suitable as epidemiological tools for population assessments (Sharma et al, 2020).

IR, as determined by clinical measurements, as above, can be determined for male and female adult and young humans.

High Male High Female

High

Adult

High High

Li, M. et al (2022), "Trends in insulin resistance: insights into mechanisms and therapeutic strategy", Signal Transduction and Targeted Therapy, Vol 7, 216. Pei, J. et al (2022), "Current studies on molecular mechanisms of insulin resistance", Journal of Diabetes Research, Vol 2022, Article ID 1863429. Sharma V.R. et al (2020), "Measuring Insulin Resistance in Humans", Hormone Research in Paediatrics, Vol 93, pp 577-588. Tsatsoulis, A. et al (2013), "Insulin resistance: An adaptive mechanism becomes maladaptive in the current environment — An evolutionary perspective", Metabolism, Vol 62, pp 622-633. AOPWiki 2023-04-03T12:14:39

2023-05-26T06:34:08

Thyroxine (T4) in serum, Decreased

T4 in serum, Decreased

Tissue

All iodothyronines are derived from the modification of tyrosine molecules (Taurog, 2000). There are two biologically active thyroid hormones (THs) in serum, triiodothyronine (T3) and T4, and a few less active iodothyronines, reverse T3 (rT3),  and 3,3'-Diiodothyronine (3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland in mammals and is the pool from which the majority of T3 in serum is generated (Zoeller et al., 2007). As such, serum T4 changes usually precede changes in other serum THs. Decreased thyroxine (T4) in serum results from one or more MIEs upstream and is considered a key biomarker of altered TH homeostasis (DeVito et al., 1999). Serum T4 is used as a biomarker of TH status because the circulatory system serves as the major transport and delivery system for TH delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In serum, it is the unbound, or ‘free’ form of the hormone that is thought to be available for transport into tissues. Free hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. There are major species differences in the predominant binding proteins and their affinities for THs (see below). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis across vertebrates (DeVito et al., 1999; Miller et al., 2009; Zoeller et al., 2007; Carr and Patiño, 2011). Normal serum T4 reference ranges can be species and lifestage specific. In rodents, serum THs are low in the fetal circulation, increasing as the fetal thyroid gland becomes functional on gestational day 17, just a few days prior to birth. After birth serum hormones increase steadily, peaking at two weeks, and falling slightly to adult levels by postnatal day 21 (Walker et al., 1980; Harris et al., 1978; Goldey et al., 1995; Lau et al., 2003). Similarly, in humans, adult reference ranges for THs do not reflect the normal ranges for children at different developmental stages, with TH concentrations highest in infants, still increased in childhood, prior to a decline to adult levels coincident with pubertal development (Corcoran et al. 1977; Kapelari et al., 2008). In some frog species, there is an analogous peak in THs in tadpoles that starts around embryonic NF stage 56, peaks at stage 62 and the declines to lower levels by stage 56 (Sternberg et al., 2011; Leloup and Buscaglia, 1977).  Additionally, ample evidence is available from studies investigating responses to inhibitors of TH synthesis in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.

Serum T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone concentrations are clinically considered more direct indicators of T4 and T3 activities in the body, but in animal studies, total T3 and T4 are typically measured. Historically, the most widely used method in toxicology is the radioimmunoassay (RIA). The method is routinely used in rodent endocrine and toxicity studies. The ELISA method is commonly used as a human clinical test method. Analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, through methods employing HPLC, liquid chromatography, immuno luminescence, and mass spectrometry are less common, but are becoming increasingly available (Hornung et al., 2015; DeVito et al., 1999; Baret and Fert, 1989; Spencer, 2013; Samanidou V.F et al., 2000; Rathmann D. et al., 2015 ). In fish early life stages most evidence for the ontogeny of thyroid hormone synthesis comes from measurements of whole body thyroid hormone levels using LC-MS techniques (Hornung et al., 2015) which are increasingly used to accurately quantify whole body thyroid hormone levels as a proxy for serum thyroid hormone levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). It is important to note that thyroid hormones concentrations can be influenced by a number of intrinsic and extrinsic factors (e.g., circadian rhythms, stress, food intake, housing, noise) (see for example, Döhler et al., 1979). Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach. This is of particular significance when assessing the very low levels of TH present in fetal serum. Detection limits of the assay must be compatible with the levels in the biological sample. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.

Taxonomic: This KE is plausibly applicable across vertebrates and the overall evidence supporting taxonomic applicability is strong. THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in zebrafish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002), and amphibian and lamprey metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). Their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of THs in the different species depends on the expression and function of specific proteins (e.g receptors or enzymes) under TH control and may vary across species and tissues. As such, extrapolation regarding TH action across species and developmental stages should be done with caution. With few exceptions, vertebrate species have circulating T4 (and T3) that are bound to transport proteins in blood. Clear species differences exist in serum transport proteins (Dohler et al., 1979; Yamauchi and Isihara, 2009). There are three major transport proteins in mammals; thyroid binding globulin (TBG), transthyretin (TTR), and albumin. In adult humans, the percent bound to these proteins is about 75, 15 and 10 percent, respectively (Schussler 2000).  In contrast, in adult rats the majority of THs are bound to TTR. Thyroid- binding proteins are developmentally regulated in rats. TBG is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). However, low levels of TBG persist into adult ages in rats and can be experimentally induced by hypothyroidism, malnutrition, or caloric restriction (Rouaze-Romet et al., 1992). While these species differences impact TH half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships of binding proteins and serum hormones during development across different species. Serum THs are still regarded as the most robust measurable key event causally linked to downstream adverse outcomes. Life stage: The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors. Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

UBERON:0001977

UBERON serum

High Female High Male

High

All life stages

High High High Moderate Moderate High High High

Axelrad DA, Baetcke K, Dockins C, Griffiths CW, Hill RN, Murphy PA, Owens N, Simon NB, Teuschler LK. Risk assessment for benefits analysis: framework for analysis of a thyroid-disrupting chemical. J Toxicol Environ Health A. 2005 68(11-12):837-55. Baret A. and Fert V.  T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay. J Biolumin Chemilumin. 1989. 4(1):149-153 Bartalena L, Robbins J. Thyroid hormone transport proteins. Clin Lab Med. 1993 Sep;13(3):583-98. Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11. Capen CC. Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol Pathol. 1997 25(1):39-48. Carr JA, Patino R. 2011. The hypothalamus-pituitary-thyroid axis in teleosts and amphibians: Endocrine disruption and its consequences to natural populations. General and Comparative Endocrinology. 170(2):299-312. Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184. Cope RB, Kacew S, Dourson M. A reproductive, developmental and neurobehavioral study following oral exposure of tetrabromobisphenol A on Sprague-Dawley rats. Toxicology. 2015 329:49-59. Corcoran JM, Eastman CJ, Carter JN, Lazarus L. (1977). Circulating thyroid hormone levels in children. Arch Dis Child. 52: 716-720. Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285. Crofton KM. Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal. 2004 Dec;24(6):1665-71. DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415. Döhler KD, Wong CC, von zur Mühlen A (1979).   The rat as model for the study of drug effects on thyroid function: consideration of methodological problems.  Pharmacol Ther B. 5:305-18. Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitaminsProc Soc Exp Biol Med. 214:302-317. Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47. Goldey ES, Crofton KM. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci. 1998 45(1):94-10 Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM.  Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Tox Appl Pharmacol. 1995 135(1):77-88. Harris AR, Fang SL, Prosky J, Braverman LE, Vagenakis AG.  Decreased outer ring monodeiodination of thyroxine and reverse triiodothyronine in the fetal and neonatal rat.  Endocrinology. 1978 Dec;103(6):2216-22 Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604. Heyland A, Hodin J. (2004). Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of non-feeding development. Evolution. 58: 524-538. Heyland A, Moroz LL. (2005). Cross-kingdom hormonal signaling: an insight from thyroid hormone functions in marine larvae. J Exp Biol. 208:4355-4361. Hill RN, Crisp TM, Hurley PM, Rosenthal SL, Singh DV. Risk assessment of thyroid follicular cell tumors.  Environ Health Perspect. 1998 Aug;106(8):447-57. Hornung MW, Kosian P, Haselman J, Korte J, Challis K, Macherla C, Nevalainen E, Degitz S (2015) In vitro, ex vivo and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol Sci 146:254-264. Hulbert AJ. Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc. 2000 Nov;75(4):519-631. Review. Kapelari K, Kirchlechner C, Högler W, Schweitzer K, Virgolini I, Moncayo R. 2008. Pediatric reference intervals for thyroid hormone levels from birth to adulthood: a retrospective study. BMC Endocr Disord. 8: 15. Lau C, Thibodeaux JR, Hanson RG, Rogers JM, Grey BE, Stanton ME, Butenhoff JL, Stevenson LA.  Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation.  Toxicol Sci. 2003 Aug;74(2):382-92. Leloup, J., and M. Buscaglia. La triiodothyronine: hormone de la métamorphose des amphibiens. CR Acad Sci 284 (1977): 2261-2263. Liu J, Liu Y, Barter RA, Klaassen CD.: Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273, 977-85, 1994 Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32. Liu YW, Chan WK. 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation. 70(1):36-45. Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220.  McClain RM. Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment. Mutat Res. 1995 Dec;333(1-2):131-42 Miller MD, Crofton KM, Rice DC, Zoeller RT.  Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 2009 117(7):1033-41 Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79. Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203. NTP National Toxicology Program.: NTP toxicology and carcinogenesis studies of 3,3'-dimethylbenzidine dihydrochloride (CAS no. 612-82-8) in F344/N rats (drinking water studies). Natl Toxicol Program Tech Rep Ser 390, 1-238, 1991. O'Connor, J. C., J. C. Cook, et al. (1998). "An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs)." Toxicol Sci 46(1): 45-60. O'Connor, J. C., L. G. Davis, et al. (2000). "Detection of dopaminergic modulators in a tier I screening battery for identifying endocrine-active compounds (EACs)." Reprod Toxicol 14(3): 193-205. Opitz R, Maquet E, Zoenen M, Dadhich R, Costagliola S. 2011. Tsh receptor function is required for normal thyroid differentiation in zebrafish. Molecular Endocrinology. 25(9):1579-1599. Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459. Rathmann D, Rijntjes E, Lietzow J, Köhrle J. (2015) Quantitative Analysis of Thyroid Hormone Metabolites in Cell Culture Samples Using LC-MS/MS. Eur Thyroid J. Sep;4(Suppl 1):51-8. Rouaze-Romet M, Savu L, Vranckx R, Bleiberg-Daniel F, Le Moullac B, Gouache P, Nunez EA. 1992. Reexpression of thyroxine-binding globulin in postweaning rats during protein or energy malnutrition. Acta Endocrinol (Copenh).127:441-448. Samanidou VF, Kourti PV. (2009) Rapid HPLC method for the simultaneous monitoring of duloxetine, venlaflaxine, fluoxetine and paroxetine in biofluids. Bioanalysis. 2009 Aug;1(5):905-17. Savu L, Vranckx R, Maya M, Gripois D, Blouquit MF, Nunez EA. 1989. Thyroxine-binding globulin and thyroxinebinding prealbumin in hypothyroid and hyperthyroid developing rats. BiochimBiophys Acta. 992:379-384. Schneider S, Kaufmann W, Strauss V, van Ravenzwaay B.    Vinclozolin: a feasibility and sensitivity study of the ILSI-HESI F1-extended one-generation rat reproduction protocol. Regul Toxicol Pharmacol. 2011 Feb;59(1):91-100. Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149. Spencer, CA. (2013). Assay of thyroid hormone and related substances. In De Groot, LJ et al. (Eds). Endotext. South Dartmouth, MA Sternberg RM, Thoemke KR, Korte JJ, Moen SM, Olson JM, Korte L, Tietge JE, Degitz SJ Jr. Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis. Gen Comp Endocrinol. 2011. 173(3):428-37 Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223. Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217. Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81Walker P, Dubois JD, Dussault JH.  Free thyroid hormone concentrations during postnatal development in the rat.  Pediatr Res. 1980 Mar;14(3):247-9. Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldúa D. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environ Sci Technol. 2011 Sep 1;45(17):7525-32. Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331. Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32. Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347. Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression. FEBS J. 2009. 276(19):5357-66. Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924. Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142. Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53 Zoeller, R. T., R. Bansal, et al. (2005). "Bisphenol-A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters RC3/neurogranin expression in the developing rat brain." Endocrinology 146(2): 607-612. AOPWiki 2016-11-29T18:41:23

2022-10-10T08:52:30

Superoxide generation, increased

Cellular

Superoxide, a reactive oxygen radical, is generated by incomplete (1-electron) reduction of molecular oxygen, O2. It is generated in, amongst other locations, the mitochondrial matrix, by the operation of the electron transport chain. Electron transport-mediated generation of superoxide is a quantitatively important route of generation. Superoxide has important cellular signalling functions but, being highly reactive, excess generation can result in damage to mitochondrial macromolecules with consequent deleterious impact on the cell (Murphy, 2009).

Mitochondrial superoxide is difficult to measure because of the high activity of superoxide dismutase in the mitochondrial matrix, which rapidly catalyses the dismutation of superoxide to O2 and hydrogen peroxide, H2O2, so measurement of mitochondrial superoxide is not routinely performed. Purified mitochondrial proteins can be manipulated so as to produce superoxide, but this is of limited physiological relevance. However, the H2O2 generated by dismutation within the mitochondrial matrix diffuses rapidly to the exterior, meaning that H2O2 generation by isolated mitochondria can be used to measure superoxide generation (Murphy, 2009).

The mitochondrial generation of superoxide is widespread in aerobic cells (Murphy, 2009).

CL:0000255

CL eukaryotic cell

High Mixed

High

Adult

High

Murphy, H.P. (2009), "How mitochondria produce reactive oxygen species", Biochemical Journal, Vol 417, pp1-13 AOPWiki 2023-04-03T12:50:24

2023-04-17T06:40:06

Thyroid hormone synthesis, Decreased

TH synthesis, Decreased

Cellular

The thyroid hormones (TH), triiodothyronine (T3) and thyroxine (T4) are thyrosine-based hormones. Synthesis of THs is regulated by thyroid-stimulating hormone (TSH) binding to its receptor and thyroidal availability of iodine via the sodium iodide symporter (NIS). Other proteins contributing to TH production in the thyroid gland, including thyroperoxidase (TPO), dual oxidase enzymes (DUOX), and the transport protein pendrin are also necessary for iodothyronine production (Zoeller et al., 2007). The production of THs in the thyroid gland and resulting serum concentrations are controlled by a negatively regulated feedback mechanism. Decreased T4 and T3 serum concentrations activates the hypothalamus-pituitary-thyroid (HPT) axis which upregulates thyroid-stimulating hormone (TSH) that acts to increase production of additional THs (Zoeller and Tan, 2007). This regulatory system includes: 1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH); 2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary; 3) hormonal transport by the plasma binding proteins; 4) cellular uptake mechanisms at the tissue level; 5) intracellular control of TH concentrations by deiodinating mechanisms; 6) transcriptional function of the nuclear TH receptor; and 7) in the fetus, the transplacental passage of T4 and T3 (Zoeller et al., 2007). TRH and the TSH primarily regulate the production of T4, often considered a “pro-hormone,” and to a lesser extent of T3, the transcriptionally active TH. Most of the hormone released from the thyroid gland into circulation is in the form of T4, while peripheral deiodination of T4 is responsible for the majority of circulating T3. Outer ring deiodination of T4 to T3 is catalyzed by the deiodinases 1 and 2 (DIO1 and DIO2), with DIO1 expressed mainly in liver and kidney, and DIO2 expressed in several tissues including the brain (Bianco et al., 2006). Conversion of T4 to T3 takes place mainly in the liver and kidney, but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009).  In mammals, most evidence for the ontogeny of TH synthesis comes from measurements of serum hormone concentrations. And, importantly, the impact of xenobiotics on fetal hormones must include the influence of the maternal compartment since a majority of fetal THs are derived from maternal blood early in fetal life, with a transition during mid-late gestation to fetal production of THs that is still supplemented by maternal THs. In humans, THs can be found in the fetus as early as gestational weeks 10-12, and concentations rise continuously until birth. At term, fetal T4 is similar to maternal levels, but T3 remains 2-3 fold lower than maternal levels. In rats, THs can be detected in the fetus as early as the second gestational week, but fetal synthesis does not start until gestational day 17 with birth at gestational day 22-23. Maternal THs continue to supplement fetal production until parturition. (see Howdeshell, 2002; Santisteban and Bernal, 2005 for review). Due to the maternal factor, the life stage specific impact of TPO inhibition after exposure to environmental chemicals is complex (Ramhoj et al., 2022). Decreased TH synthesis in the thyroid gland may result from several possible molecular-initiating events (MIEs) including: 1) Disruption of key catalytic enzymes or cofactors needed for TH synthesis, including TPO, NIS, or dietary iodine insufficiency. Theoretically, decreased synthesis of Tg could also affect TH production (Kessler et al., 2008; Yi et al., 1997). Mutations in genes that encode requisite proteins in the thyroid may also lead to impaired TH synthesis, including mutations in pendrin associated with Pendred Syndrome (Dossena et al., 2011), mutations in TPO and Tg (Huang and Jap 2015), and mutations in NIS (Spitzweg and Morris, 2010). 2) Decreased TH synthesis in cases of clinical hypothyroidism may be due to Hashimoto's thyroiditis or other forms of thyroiditis, or physical destruction of the thyroid gland as in radioablation or surgical treatment of thyroid lymphoma. 3) It is possible that TH synthesis may also be reduced subsequent to disruption of the negative feedback mechanism governing TH homeostasis, e.g. pituitary gland dysfunction may result in a decreased TSH signal with concomitant T3 and T4 decreases. 4) More rarely, hypothalamic dysfunction can result in decreased TH synthesis.  Increased fetal TH levels are also possible. Maternal Graves disease, which results in fetal thyrotoxicosis (hyperthyroidism and increased serum T4 levels), has been successfully treated by maternal administration of TPO inhibitors (c.f., Sato et al., 2014).   It should be noted that different species and different life stages store different amounts of TH precursors and iodine within the thyroid gland. Thus, decreased TH synthesis via transient iodine insufficiency or inhibition of TPO may not affect TH release from the thyroid gland until depletion of stored iodinated Tg. Adult humans may store sufficient Tg-DIT residues to serve for several months to a year of TH demand (Greer et al., 2002; Zoeller, 2004). Neonates and infants have a much more limited supply of less than a week. While the TH system is highly conserved across vertebrates, there are some taxon-specific considerations. Zebrafish and fathead minnows are oviparous fish species in which maternal THs are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic TH synthesis is initiated. Maternal transfer of THs to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016). Decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. The components of the TH system responsible for TH synthesis are highly conserved across vertebrates and therefore interference with the same molecular targets compared to mammals can lead to decreased TH synthesis (TPO, NIS, etc.) in fish. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for these genes is also maternally transferred and increasing expression of most transcripts during hatching and embryo-larval transition indicates a fully functional HPT axis in larvae (Vergauwen et al., 2018). Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus TH synthesis compared to TSH-releasing hormone (TRH). Also, in most fish species thyroid follicles are more diffusely located in the pharyngeal region rather than encapsulated in a gland.

Decreased TH synthesis is often implied by measurement of TPO and NIS inhibition measured clinically and in laboratory models as these enzymes are essential for TH synthesis. Rarely is decreased TH synthesis measured directly, but rather the impact of chemicals on the quantity of T4 produced in the thyroid gland, or the amount of T4 present in serum is used as a marker of decreased T4 release from the thyroid gland (e.g., Romaldini et al., 1988). Methods used to assess TH synthesis include, incorporation of radiolabeled tracer compounds, radioimmunoassay, ELISA, and analytical detection.    Recently, amphibian thyroid explant cultures have been used to demonstrate direct effects of chemicals on TH synthesis, as this model contains all necessary synthesis enzymes including TPO and NIS (Hornung et al., 2010). For this work THs was measured by HPLC/ICP-mass spectometry. Decreased TH synthesis and release, using T4 release as the endpoint, has been shown for thiouracil antihyperthyroidism drugs including MMI, PTU, and the NIS inhibitor perchlorate (Hornung et al., 2010). Techniques for in vivo analysis of TH system disruption among other drug-related effects in fish were reviewed by Raldua and Piña (2014). TIQDT (Thyroxine-immunofluorescence quantitative disruption test) is a method that provides an immunofluorescent based estimate of thyroxine in the gland of zebrafish (Raldua and Babin, 2009; Thienpont et al., 2011; Jomaa et al., 2014; Rehberger et al., 2018).  Thienpont used this method with ~25 xenobiotics (e.g., amitrole, perchlorate, methimazole, PTU, DDT, PCBs). The method detected changes for all chemicals known to directly impact TH synthesis in the thyroid gland (e.g., NIS and TPO inhibitors), but not those that upregulate hepatic catabolism of T4. Rehberger et al. (2018) updated the method to enable simultaneous semi-quantitative visualization of intrafollicular T3 and T4 levels. Most often, whole body TH level measurements in fish early life stages are used as indirect evidence of decreased TH synthesis (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). Analytical determination of TH levels by LC-MS is becoming increasingly available (Hornung et al., 2015). More recently, transgenic zebrafish with fluorescent thyroid follicles are being used to visualize the compensatory proliferation of the thyroid follicles following inhibition of TH synthesis among others (Opitz et al., 2012).

Taxonomic: This KE is plausibly applicable across vertebrates. Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across vertebrate taxa, with in vivo evidence from humans, rats, amphibians, some fish species, and birds, and in vitro evidence from rat and porcine microsomes. Indeed, TPO and NIS mutations result in congenital hypothyroidism in humans (Bakker et al., 2000; Spitzweg and Morris, 2010), demonstrating the essentiality of TPO and NIS function toward maintaining euthyroid status. Though decreased serum T4 is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis, clinical and veterinary management of hyperthyroidism and Graves’ disease using propylthiouracil and methimazole, known to decrease TH synthesis, indicates strong evidence for chemical inhibition of TPO (Zoeller and Crofton, 2005). Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish (e.g., fathead minnow, zebrafish) rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). In externally developing fish species, decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. TPO inhibition in a homozygous knockout line abolished the T4 production in thyroid follicles of mutant zebrafish with phenotypic abnormalities occurring from 20 dpf onwards but not before 10 dpf (Fang et al., 2022). Therefore, it is still uncertain when exactly embryonic TH synthesis is activated and thus when exactly this process becomes sensitive to disruption. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It currently remains unclear when exactly embryonic TH production is initiated in zebrafish. Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

UBERON:0002046

UBERON thyroid gland

CL:0002258

CL thyroid follicular cell

High Male High Female

High

All life stages

High High Moderate High Moderate High

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2022-11-04T09:25:39

Increase, Oxidative Stress

Molecular

“Oxidative stress is traditionally defined as an imbalance between production of ROS and endogenous antioxidants” (Small et al., 2018). Reactive Oxygen Species (ROS), the core perpetrators of oxidative stress, are highly reactive free radical oxygen molecules (Turrens, 2003; Valko et al., 2005; Hancock et al., 2001). These free radicals are strong oxidants that in high amounts, could irreversibly damage the cell and its organelles. ROS, however, are not by default detrimental to cellular function but contribute to normal activities, under physiological conditions. Reactive oxygen species can behave as signaling molecules by oxidizing proteins, lipids, and polynucleotides (Zhao et al., 2019; Hancock et al., 2001; Gorlach et al., 2015). When present in tolerable amounts, superoxides (O2-) behave as signalling molecules important in cell proliferation, hypoxia adaption, and cell fate determination but when present in excess or unregulated, induce cell damage and death (Zhao et al., 2019). Under normal oxidative phosphorylation, approximately 1-2% of the oxygen reduced by mitochondria converts into reactive oxygen species (ROS) at intermediate steps of the respiratory chain, during electron transfer (Kowaltowski and Vercesi, 1998; Volka et al., 2005; Li et al., 2003). On top of ROS generation occurring during natural cell metabolism, several other mitochondrial enzymes, such as cytochrome P450, are linked to ROS production and, interestingly many of these systems are modulated by calcium (Gorlach et al., 2015; Hancock et al., 2001). The main reasons that these antioxidant systems are in place are to regulate the constant, regular production of ROS and their signalling functionality. Oxidative stress is defined as a disruption of the pro-oxidant-antioxidant balance, favouring pro-oxidants, potentially damaging cellular components (Sies, 1991). It is important to note the fact that oxidative stress is generally caused by one of three reasons: either there is a significant increase in ROS; or there is a significant decrease in antioxidant function; or the two factors working in tandem. This distinction is crucial in determining methods of assessment of oxidative stress. Other than mitochondrial ROS production, one of the primary producers of superoxide (O2-) is NADPH, doing so by a single electron reduction of molecular oxygen (Panday et al., 2014). NADPH oxidase, is a cytosolic-membrane protein, implicated in host defence, cellular signalling, and gene expression regulation. It is thought to produce ROS partly for defence against microbial pathogens (Panday et al., 2014). Due to its volatile nature, once formed, O2‑ released into the inter-membrane space (IMS) by CIII inevitably generates hydrogen peroxide (H2O2), a reaction is catalyzed by superoxide dismutase (SOD) (Hancock et al., 2001). Once present, the fate of H2O2 is variable and its reduction to H2O can be catalyzed by glutathione peroxidase (GPx) or peroxisomal catalase. Otherwise, it can be further converted into highly reactive hydroxyl radicals, particularly in the presence of metal ions (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Under normal mitochondrial conditions, antioxidant enzymes and molecules – such as, superoxide dismutase, glutathione peroxidase, catalase, and glutathione – balance ROS generation by scavenging or binding to ROS (Santos et al., 2007). ROS disposal involves superoxide dismutase (SOD) converting O2- to H2O2, then detoxified into H2O by glutathione and catalase (Xu & Møller, 2010). Also, in case of mitochondrial damage or dysfunction, the antioxidant defence system may be impaired and its capacity drops. The resultant imbalance of the production and removal of ROS is oxidative stress. Due to the nature of oxidative stress induction and the short half-life of ROS, measuring oxidative stress through antioxidant function is a more accurate and effective way to determine the state of the cell. Oxidative stress behaves as a positive feedback loop, leading to the over-production of free-radicals as ROS further damage the mitochondria and antioxidant capacity continues to decrease (Hancock et al., 2001; Turrens, 2003; Valko et al., 2005). Antioxidant defenses can also be inhibited, further contributing to mitochondrial dysfunction; this is commonly induced by exposure to heavy metals (Blajszczak and Bonini, 2017). Due to the formation of this positive feedback loop, we experience a gap in knowledge as to which event initiated the cycle; was the increased ROS production the cause of mitochondrial dysfunction or did the mitochondrial dysfunction lead to increased ROS production? <div>Heavy metal exposure in aerobic organisms increases ROS formation through redox cycling, where metals with different valence states (for example Fe, Cu, and Cr) directly produce ROS as they are reduced by cellular antioxidants and then react with oxygen (Shaki et al., 2012; Shaki et al., 2013; Pourahmad et al., 2006; Santos et al., 2007). The production of highly reactive hydroxyl radicals under mitochondrial oxidative stress in the presence of transition metals occurs via the Fenton reaction or Haber-Weiss reaction (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Metals have been shown to inhibit ROS-detoxifying enzymes. By binding to these enzymes, metals can inhibit their antioxidant functions, and cause an accumulation of ROS and increased synthesis of more antioxidants in order to combat the oxidative stress (Blajszczak and Bonini, 2017).</div>

<table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <tbody> <tr> <td>Assay Type & Measured Content</td> <td>Description</td> <td>Dose Range Studied</td> <td> Assay Characteristics (Length / Ease of use/Accuracy) </td> </tr> <tr> <td> ROS Formation in the Mitochondria assay Measuring (Shaki et al., 2012) </td> <td>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.” (Shaki et al., 2012)</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.” (Shaki et al., 2012)</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.” (Heyno et al., 2008) </td> <td> 0, 10, 30  μM Cd2+ 2  μM antimycin A</td> <td> </td> </tr> <tr> <td> Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997)</td> <td>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)” </td> <td> </td> <td> Strong/easy medium</td> </tr> <tr> <td> DCFH-DA Assay Detection of hydrogen peroxide production (Yuan et al., 2016) </td> <td> Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production.    </td> <td>0-400 µM</td> <td> Long/ Easy High accuracy </td> </tr> <tr> <td> H2-DCF-DA Assay Detection of superoxide production (Thiebault et al., 2007) </td> <td>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.</td> <td>0–600 µM</td> <td> Long/ Easy High accuracy </td> </tr> <tr> <td>CM-H2DCFDA Assay</td> <td>**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**</td> <td> </td> <td> </td> </tr> </tbody> </table>

Oxidative stress can happen in all forms of life.

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Mitochondrial electron transport chain in heavy metal-induced neurotoxicity : Effects of cadmium , mercury , and copper. Thescientificworld, 2012, 1-14. doi:10.1100/2012/136063 Bhadauria, S., & Flora, S. J. S. (2007). Response of arsenic-induced oxidative stress, DNA damage, and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats. Cell Biol Toxicol, 23, 91-104. doi:10.1007/s10565-006-0135-8 Blajszczak, C., & Bonini, M. G. (2017). Mitochondria targeting by environmental stressors : Implications for redox cellular signaling. Toxicology, 391, 84-89. doi:10.1016/j.tox.2017.07.013 Buelna-Chontal, M., Franco, M., Hernandez-Esquivel, L., Pavon, N., Rodriguez-Zalvala, J. S., Correa, F., . . . Chavez, E. (2017). CDP-choline circumvents mercury-induced mitochondrial damage and renal dysfunction. Cell Biology International, 41, 1356-1366. doi:10.1002/cbin.10871 Chtourou, Y., Garoui, E. m., Boudawara, T., & Zeghal, N. (2014). Protective role of silymarin against manganese-induced nephrotoxicity and oxidative stress in rat. Environ Toxicol, 29, 1147-1154. doi:10.1002/tox.21845 Ferreira, G. K., Cardoso, E., Vuolo, F. S., Michels, M., Zanoni, E. T., Carvalho-Silva, M., . . . Paula, M. M. S. (2015). Gold nanoparticles alter parameters of oxidative stress and energy metabolism in organs of adult rats. Biochem. Cell Biol., 93, 548-557. doi:10.1139/bcb-2015-0030 Görlach, A., Bertram, K., Hudecova, S., & Krizanova, O. (2015). Calcium and ROS: A mutual interplay. Redox Biology, 6, 260-271. doi:doi:10.1016/j.redox.2015.08.010 Hancock, J. T., Desikan, R., & Neill, S. J. (2001). Role of reactive oxygen species in cell signalling pathways. Biochemical Society Transactions, 29(Pt 2), 345-350. doi:10.1042/0300-5127:0290345 [doi] Hao, Y., Huang, J., Liu, C., Li, H., Liu, J., Zeng, Y., . . . Li, R. (2016). Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity. Scientific Reports, doi:10.1038/srep38942 Heyno, E., Klose, C., & Krieger-Liszkay, A. (2008). Origin of cadmium-induced reactive oxygen species production: Mitochondrial electron transfer versus plasma membrane NADPH oxidase. New Phytologist, 179, 687-699. doi:10.1111/j.1469-8137.2008.02512.x Huerta-García, E., Perez-Arizti, J. A., Marquez-Ramirez, S. G., Delgado-Buenrostro, N. L., Chirino, Y. I., Iglesias, G. G., & Lopez-Marure, R. (2014). Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells. Free Radical Biology and Medicine, 73, 84-94. doi:10.1016/j.freeradbiomed.2014.04.026 Jozefczak, M., Bohler, S., Schat, H., Horemans, N., Guisez, Y., Remans, T., . . . Cuypers, A. (2015). Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium. Annals of Botany, 116(4), 601-612. doi:10.1093/aob/mcv075 Kehrer, J. P. (2000). The Haber–Weiss reaction and mechanisms of toxicity. Toxicology, 149(1), 43-50. doi:<a href="https://doi.org/10.1016/S0300-483X(00)00231-6" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1016/S0300-483X(00)00231-6</a> Kharroubi, W., Dhibi, M., Mekni, M., Haouas, Z., Chreif, I., Neffati, F., . . . Sakly, R. (2014). Sodium arsenate induce changes in fatty acids profiles and oxidative damage in kidney of rats. Environ Sci Pollut Res, 21, 12040-12049. doi:10.1007/s11356-014-3142-y Kowaltowski, A. J., & Vercesi, A. E. (1999). Mitochondrial damage induced by conditions of oxidative stress. Free Radical Biology and Medicine, 26(3), 463-471. doi:<a href="https://doi.org/10.1016/S0891-5849(98)00216-0" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1016/S0891-5849(98)00216-0</a> Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649. Li, N., Ragheb, K., Lawler, G., Sturgis, J., Melendez, J. A., & Robinson, J. P. (2003). Mitochondrial complex I inhibitor rotenone induced apoptosis through enhancing mitochondrial reactive oxygen species production. The Journal of Biological Chemistry, 278(10), 8516-8525. Liang, Shih-Shin & Shiue, Yow-Ling & Kuo, Chao Jen & Guo, Su-Er & Liao, Wei-Ting & Tsai, Eing. (2013). Online Monitoring Oxidative Products and Metabolites of Nicotine by Free Radicals Generation with Fenton Reaction in Tandem Mass Spectrometry. TheScientificWorldJournal. 2013. 189162. 10.1155/2013/189162. Liu, S., Xu, L., Zhang, T., Ren, G., & Yang, Z. (2010). Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. Toxicology, 267, 172-177. doi:10.1016/j.tox.2009.11.012 Lunyera, J., & Smith, S. R. (2017). Heavy metal nephropathy: Considerations for exposure analysis. Kidney International, 92, 548-550. doi:http://dx.doi.org/10.1016/j.kint.2017.04.043 Miyayama, T., Arai, Y., Suzuki, N., & Hirano, S. (2013). Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells follwoing exposure to silver nitrate. Toxicology, 305, 20-29. doi:10.1016/j.tox.2013.01.004 Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466 Panday, A., Sahoo, M., Osorio, D., Barta, S. (2014). NADPH oxidases: an overview from structure to innate immunity-associated pathologies. Cell Mol Immunol 12, 5–23 (2015). <a href="https://doi.org/10.1038/cmi.2014.89" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/cmi.2014.89</a> Pourahmad, J., Ghashang, M., Ettehadi, H. A., & Ghalandari, R. (2006). A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity. Environmental Toxicology, 21(4), 349-354. doi:10.1002/tox.20196 Sabath, E., & Robles-Osorio, M. L. (2012). Renal health and the environment: Heavy metal nephrotoxicity. Revista Nefrologia, doi:10.3265/Nefrologia.pre2012.Jan.10928 Santos, N. A. G., Catão, C. S., Martins, N. M., Curti, C., Bianchi, M. L. P., & Santos, A. C. (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Archives of Toxicology, 81(7), 495-504. doi:10.1007/s00204-006-0173-2 Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015 Shaki, F., Hosseini, M., Ghazi-Khansari, M., & Pourahmad, J. (2013). Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria. Metallomics, 5(6), 736-744. doi:10.1039/c3mt00019b Sies, H. (Ed.), Oxidative Stress, Oxidants and Antioxidants, Academic Press, San Diego, CA (1991), pp. XV-XXII Small, D. M., Sanchez, W. Y., Roy, S. F., Morais, C., Brooks, H. L., Coombes, J. S., . . . Gobe, G. (2018). N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses. American Physiological Society - Renal Physiology, 314, F956-F968. doi:10.1152/ajprenal.00057.2017 Thiébault, C., Carrière, M., Milgram, S., Simon, A., Avoscan, L., & Gouget, B. (2007). Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells. Toxicological Sciences : An Official Journal of the Society of Toxicology, 98(2), 479-487. doi:kfm130 [pii] Turk, E., Kandemir, F. M., Yildirim, S., Caglayan, C., Kucukler, S., & Kuzu, M. (2019). Protective effect of hesperidin on sodium arsenite-induced nephrotoxicity and hepatotoxicity in rats. Biological Trace Element Research, 189, 95-108. doi:10.1007/s12011-018-1443-6 Turrens, J. F. (2003). Mitochondrial formation of reactive oxygen species. The Journal of Physiology, 552(Pt 2), 335-344. doi:jphysiol.2003.049478 [pii] Tyagi, R., Rana, P., Gupta, M., Khan, A. R., Bhatnagar, D., Bhalla, P. J. S., . . . Kushu, S. (2011). Differntial biochemical response of rat kidney towards low and high doses of NiCl2 as revealed by NMR spectroscopy. Journal of Applied Toxicology, 33, 134-141. doi:10.1002/jat.1730 Valko, M., Morris, H., & Cronin, M. T. (2005). Metals, toxicity and oxidative stress. Current Medicinal Chemistry, 12(10), 1161-1208. doi:10.2174/0929867053764635 [doi] Wang, L., Li, J., Li, J., & Liu, Z. (2009). Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1 Xu, X. M., & Moller, G. S. (2010). ROS removal by DJ-1. Plant Signaling & Behaviour, 5(8), 1034-1036. doi:10.4161/psb.5.8.12298 Yeh, Y., Lee, Y., Hsieh, Y., & Hwang, D. (2011). Dietary taurine reduces zinc-induced toxicity in male wistar rats. Journal of Food Science, 76(4), 90-98. doi:10.1111/j.1750-3841.2011.02110.x Yuan, Y., Zheng, J., Zhao, T., Tang, X., & Hu, N. (2016). Uranium-induced rat kidney cell cytotoxicity is mediated by decreased endogenous hydrogen sulfide (H2S) generation involved in reduced Nrf2 levels. Toxicology Research, 5(2), 660-673. doi:10.1039/C5TX00432B Zhao, R., Jiang, S., Zhang, L., & Yu, Z. (2019). Mitochondrial electron transport chain, ROS generation and uncoupling (review). International Journal of Molecular Medicine, 44(1), 3-15. doi:10.3892/ijmm.2019.4188 AOPWiki 2022-02-04T13:55:38

2022-03-03T10:40:06

<upstream-id>8c6a0578-8f22-43c2-8d08-30db13741044</upstream-id> <downstream-id>1713a78c-2579-4feb-8caf-3f0f8d7c9e5d</downstream-id> Succinate dehydrogenase (SDH) is a 4 subunit mitochondrial enzyme that oxidises succinate to fumarate, with the corresponding reduction of FAD to FADH2. FADH2 is subsequently reoxidised by transfer of electrons to the mitochondrial electron transfer chain (ETC). SDH thus links the tricarboxylic acid (TCA) cycle to mitochondrial electron transfer. It is the only TCA cycle enzyme to be embedded in the mitochondrial inner membrane, as opposed to located free in the mitochondrial matrix, comprising complex II of the ETC. The mitochondrial ETC is the largest source of superoxide radicals in most mammalian cells, complex II being one source of those radicals. Inhibition of mammalian complex II activity has been recorded for many chemicals, including fungicides, other environmental chemicals and pharmaceuticals, being associated with an increase in superoxide generation in some instances. Because of the size and complexity of complex II, with multipled potential binding sites for inhibitors, generation of superoxide radical is not, per se, a guaranteed outcome of inhibition of succinate oxidation. The site and nature of the enzyme-inhibitor interaction is potential determining factor. This KER is concerned with the direct reversible or irreversible binding of inhibitory chemicals to complex II, leading to a reduction in the dehydrogenation of succinate, and the potential for consequent generation of mitochondrial superoxide radicals. Other effects, such as reduction in expression of complex II components, are not covered.

The following search was executed in Pubmed on 27/04/2023: ("complex II"[Title] and inhibit* and mitochond*) OR ("succinate dehydrogenase" AND inhibition) generating 2341 hits. In order to reduce the hits to a more tractable set a further specification of "superoxide" was added to the second bracketed term ("complex II"[Title] and inhibit* and mitochond*) OR ("succinate dehydrogenase" AND inhibition AND superoxide) generating 286 hits whose abstracts were reviewed for further reference to direct chemical inhibition of SDH or complex II activity. As the search term contains "superoxide", some hits from this search also included information about potential impact on superoxide generation. Several articles provided evidence for multiple interactions by a chemical that could also potentially give rise to increase in superoxide concentrations (e.g. interactions at both ETC complexes II and III). Such articles have not been cited as supporting evidence for this KER. In those articles that have been cited in support of this KER, the lack of evidence presented for additional interactions, other than SDH inhibition, that could potentially give rise to increased superoxide production does not rule out the possibility of such interactions being observed in the future.

Evidence is provided in terms of (i) the biological plausibility of the KER, and (ii) empirical evidence that supports, quantitatively or qualitatively, the manifestation of the relationship in mammals in vivo, or in mammalian ex vivo or in vitro systems.

<ol> <li>multiple inhibitors of SDH/mitochondrial complex II are known,</li> <li>interference with the mitochondrial ETC is widely known to result in generation of excess superoxide,</li> <li>complex II is known to be able to generate superoxide radical under certain conditions.</li> </ol> Consequently, inhibition of SDH/complex II is potentially liable to alter the production of superoxide radicals.

Numerous instances of chemicals both inhibiting SDH/complex II and generating excess mitochondrial superoxide have been reported. 2-thenoyltrifluoroacetone (TTFA) and the anticancer agent lonidamine have been shown to inhibit succinate dehydrogenation to fumarate in a variety of cell lines (DB-1, HepG2, HCT116 and HeLa), and to inhibit the generation of fumarate and malate (into which fumarate is converted by the TCA cycle enzyme fumarase) in isolated mouse liver mitochondria.  Both compounds additionally lead to an increase in reactive oxygen (ROS) species generation in DM-1 cells (Guo et al, 2016). Similarly, trans-4,5-dihydroxy-2-cyclopentene-1-one (DCHP), derived from heat-treated citrus pectin inhibits complex II, in HCT116 cells, and leads to an increase in ROS generation in mitochondria isolated from HCT-116 cells (Chen et al, 2021).

Succinate dehydrogenase is a large, multi-protein, multi-centre enzyme, with separate binding sites for succinate (which is oxidised to fumarate) and ubiquinone (which is reduced to ubiquinol) in the two part-reactions that together comprise the full reaction (succinate + ubiquinone -> fumarate + ubiquinol). Electrons can be released at different sites for the reduction of oxygen to superoxide, so the relationship between inhibition of two-electron succinate oxidation, inhibition of two-electron ubiquinone reduction, and stiumlation of one-electron reduction of oxygen to superoxide depends on the particular inhibition mechanism and characteristics of a specific inhibitor. Thus, 3-nitroproprionate (3-NPA), a structural analogue of succinate, inhibits the succinate oxidation step, but does not generate ROS (Guo et al, 2016), so there is a complete disconnect between inhibition of the enzyme and generation of superoxide for this compound. For compounds that both inhibit the reaction, and generate superoxide, a quantitative linkage has to be defined in terms of: <ol> <li>On the one hand the rate of superoxide generation, and</li> <li>On the either hand: <ol> <li>The extent of inhibition of succinate oxidation, and/or</li> <li>The extent of inhibition of ubiquinone reduction.</li> </ol> </li> </ol> These relationships may not be quantitatively equal.

Reversible or irreversible binding to, and inhibition of, succinate dehydrogenase potentially occurs within seconds or minutes of exposure in in vitro or in vivo systems. In the presence of continued inhibition, superoxide concentrations build up in respiring mitochondria or cells occurs over a period of minutes to hours. The consequent downstream increases in intracellular superoxide generation can have multiple impacts on affected cells, leading to inhibition of susceptible enzymes, changes in enzyme expression levels and apoptosis. These changes can occur within minutes to hours of exposure. Thus, separating the proximal manifestation of the KER - the production of superoxide - from the distal manifestation of consequent events - is limited to a period of seconds to minutes for practical interpretation, relatively free of the downstream consequences. ROS generation in DB-1 cells has been observed during 4 h incubations with lonidamine and with TTFA (Guo et al, 2016).

The simultaneous inibition of the reduction of succinate to fumarate, and the generation of ROS has been demonstrated in isolated mouse liver mitochondria (Guo et al, 2016)

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b4313760300> AOPWiki 2023-04-03T12:51:59

2023-05-04T07:10:23

<upstream-id>1713a78c-2579-4feb-8caf-3f0f8d7c9e5d</upstream-id> <downstream-id>ae48a321-4090-4f7a-bd4e-bf23611ebf1a</downstream-id> Superoxide, a reactive oxygen radical is produced by one-electron reduction of molecular oxygen by multiple intracellular enzymes. Under normal physiological conditions, superoxide generation and breakdown are held in balance by the enzymatic and non-enzymatic components of the cell, including superoxide dismutase, with the result that dangerous levels of superoxide are not produced; under conditions in which superoxide production exceeds the ability of the cell to catabolise it, reactive oxygen molecules can accumulate, resulting in oxidative damage to cell components, a condition known as oxidative stress.

The following pubmed search was performed on 30/05/2023: "superoxide"[Title] and "oxidative stress"[Title] AND mitochondri*[Title] which generated 37 hits. The abstracts were read to identify articles that appeared relevant to the development of this KER, resulting in 12 being selected for study.

This KER is supported both by its biological plausibility and by empirical data.

Superoxide (O2-.) is produced by one-electron reduction of molecular oxygen (O2) by multiple intracellular enzymes, including xanthine oxidase, nitric oxide synthase, aldehyde oxidase, NADPH oxidases, fumarase, and complexes within the mitochondrial electron transport chain, the latter source being quantitatively the most important in most tissues. Superoxide dismutase isozymes in the mitochondria, cytoplasm and extracellular fluid catalyse the disproportionation of superoxide to molecular oxygen and hydrogen peroxide (H2O2): 2O2-. + 2H+ -> H2O2 + O2 Hydogen peroxide can be catabolised by catalase or glutathione peroxidase, reducing the risk of reactions involving the peroxide ion (O22-). Superoxide can also generate the highly reactive hydroxyl radical (HO.) by Fe2+-catalysed reaction with hydrogen peroxide: O2-. + H2O2  -> HO. + O2 and peroxynitrite (ONOO.), a strong oxidant, by reaction with nitric oxide (NO.): O2-. + NO.  -> ONOO. Under normal physiological conditions, superoxide generation and breakdown are held in balance by the enzymatic and non-enzymatic (such as glutathione) components of the cell's antioxidant capability. Under conditions, however, in which superoxide production exceeds the ability of the cell to catabolise it, reactive oxygen molecules can accumulate with detrimental impact on the cell components, a condition known as oxidative stress. See Indo et al (2015) for a review.

<ol> <li>Treatment with 25-250ug/mL calcium oxalate monohydrate increased the rate of superoxide generation from mitochondria of permeabilised renal epithelial cells (LLC-PK1 and MDCK), and led to a decrease in both total and reduced glutathione content (Khand et al, 2001).</li> <li> sod2-/- (mitochondrial SOD) mice treated with antioxidants EUK-8, EUK134 and EU-189 show reduced lethal and neurodegenerative effects of excess superoxide generation (Hinderfield et al, 2004). </li> <li> Overexpression of MnSOD in diabetic mice prevented: (i) the diabetes-induced decreases in retinal glutathione concentration and total antioxidant capacity, and (ii) the diabetes-induced increases in the levels of 8-hydroxy 2'-deoxyguanosine and nitrotyrosine, all markers of oxidative stress (Kowluru et al, 2006). </li> </ol> Further evidence is cited in Indo et al (2015).

The quantiative understanding of this KER is low

High Unspecific

High

Adult

High

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b43137b2b28> AOPWiki 2023-04-24T04:20:03

2023-06-01T17:09:43

<upstream-id>ae48a321-4090-4f7a-bd4e-bf23611ebf1a</upstream-id> <downstream-id>782c23bc-2272-45d6-8660-ab7da297c85e</downstream-id> Increases in oxidative stress in the thyroid gland in vivo and in thyroid cells or cell lines in vitro have been shown to cause detrimental change to multiple aspects of thyroid structure and function. Within this KER, evidence is collated that such changes could include reduction in thyroxine (T4) synthesis, with consequential reduction in T4 secretion into the blood.

A search of Pubmed was made for the following terms: ((thyroxine[Title] OR thyroid[Title]) AND "oxidative stress"[Title]) OR (thyroxine[Title/Abstract] AND ROS[Title/Abstract]) that retrieved 194 hits on 10/05/2023. The abstracts of these hits were individually inspected for any indication of reference to data relevant to the impact of oxidative stress on thyroid hormone synthesis/release (whether stimuatory, inhibitive or without effect). This inspection resulted in the identification of 33 publications for initial detailed investigation. These publications were reviewed in full, along with any citations within them that indicated further information regarding this KER - supportive or otherwise - could be found within them.

Increases in oxidative stress in the thyroid gland in vivo and in thyroid cells or cell lines in vitro have been shown to cause detrimental change to multiple aspects of thyroid structure and function, as is observed in a wide range of organs, tissues and cells or cell lines, and are implicaated in the aetiology of numerous thyroid disorders, including the development of thyroid nodules, Hashimoto's thyroiditis, Graves disease and thyroid cancer (see Kochman et al (2021) and Macvanin et al (2023) for overviews). Given that one of the major functions of the thyroid is to generate thyroxine (along with its structural analogue triiodothyronine (T3)), it is plausible that cellular dysfunction can lead to a reduction in the synthesis of T4 and T3.

Male albino rats treated with 3mg/kg carbon tetrachloride (CCL4) intraperitonally (ip) on two occasions 3 days apart showed significantly reduced serum triiodithyronine (T3) and thyroxine (T4) and increase in TSH. Thyroid weight, and relative thyroid weight increased, as did thyroidal DNA damage. Follicular colloid was depleted, blood thyroidal blood vessels were congested and follicular cells showed hyperplasia. Thyroidal glutathione (GSH) decreased, but thyroidal TBARS (thiobarbituric acid reactive substances), hydrogen peroxide (H2O2) and nitrites increased. Protein content of thyroid 12000g supernatant decreased. Catalase and peroxidase activities and superoxide dismutase specific activities were significantly reduced, as were the specific activities of glutathione transferase, glutathione reductase, glutathione peroxidase and quinone reductase. The specific activity of gamma-glutamyl transferase, an enzyme of the glutathione cycle, involved in the synthesis of glutathione, was increased. The decrease in glutathione, and the increases in TBARS and H2O2 demonstrate an increase in oxidative stress, supported by the observation of increased DNA damage. The enzyme changes observed could be contributory factors in that oxidative stress increase.  The histological observations indicate significant changes in thyroidal form and function, potentially leading to a decrease in net T4 synthesis and secretion. All observed and measured changes were mitigated by administration of a methanolic extract of Sonchus asper, an effect hypothesised to be mediated by anti-oxidant compounds (sesquiterpene lactone alkaloids, ascorbic acid and carotenoids) in the plant. Most effects were mitigated in a dose-dependent manner (using 100 or 200mg/kg of extract administered orally). Sonchus asper extract given alone did not significantly effect any of the biochemical measurements or histological observations (Khan 2012). Male Sprague-Dawley rats treated with DEHP (250, 500, 750mg/kg/day)for 30 days showed a decrease in plasma total free and total triiodothyronine (fT3 and tT3), free and total thyroxine (fT4 and tT4) and thyrotropin releasing hormone (TRH), with unchanged levels of plasma TSH. Histological assessment of the thyroid showed dose-dependent increases in the number of follicular cells and reduction in the follicular cavity volume. Ultrastructural changes observed with TEM included changes to nucleus, dilation of the RER and appearance of vacuoles. Reactive oxygen species (ROS) were generated in the liver. In vitro, Nthy-ori 3-1 cells treated with 400µM for 24hours showed an increase in ROS generation over controls that was largely diminished by the presence of 1mM N-acetyl cycteine, a ROS scavenger (Ye et al, 2017). Male Wistar rats treated with the synthetic pyrethroid insecticide lambda-cyhalothrin (α-cyano-3-phenoxy -benzyl-3-(2-chloro-3, 3,3-trifluoro-1-propenyl)-2, 2-dimethylcyclopropanecarboxylate, LCT) at 0.79mg/kg (1% of the LD50) for 3 days/week for 4 weeks for a total study duration of 30 days showed significant decreases in plasma T3 and T4 and a significant increase in TSH. Investigations of the thyroid in treated rats showed multiple structural, biochemical and pathological alterations, including degeneration of thyroid follicles; reduction in periodic acid-Schiff staining in colloid and mercury bromophenol blue staining in colloid and follicular cells indicating, respectively, reduction in carbohydrate-containing macromolecules and protein content; reduction of mean follicular diameter from 35µm to 23µm; statistically significant increases in measures of DNA damage (tail length and percent of DNA damaged) determined in the comet assay. Statistical increases in plasma malondialdehyde (MDA, a marker of reactive oxygen species (ROS) activity), superoxide dismutase and catalase indicate an increase in the body burden of ROS caused by LCT treatment (Al-Amoudi et al, 2018). All of these changes were ameliorated by co-administration of ginger extract with LCT, an effect ascribed to the anti-oxidant propeties of compounds in the ginger extract. Male albino rats treated with 200mg/kg/day bisphenol A (BPA) orally for 35 days showed significant increase in serum TSH and decrease in T3 and T4 compared to control animals. Thyroid weight, but not body weight was significantly reduced. Markers of increased oxidative stress or damage in the thyroid include inreased malondialdehyde concentration, DNA damage (increased tail length and moment, and increased % damaged DNA in the comet assay) and decreased reduced glutathione concentration. Thyroidal gene or protein expression changes included increased induction of myeloperoxidase, decrease in nuclear factor erythroid-derived 2-like 2 (Nrf-2) gene expression and protein concentration and haem-oxygenase 1 (HO-1) expression, and increase in inducible nitric oxide synthase (iNOS) compared to control. mRNA levels of TSHR, TPO and NIS were all reduced. Histological evaluation of the thyroid revealed BPA-induced congestion of capillaries, increased lymphocyte infiltration, increase in variability of follicle size and marked decrease in colloid content compared to control animals. Nearly all effects were wholly or partially ameliorated by co-administration of ginger extract (Mohammed et al, 2020). Male Wistar rates exposed to PCB118 for 13 weeks showed a dose-dependent decrease in plasma free triiodothyronine (fT3), plasma free thyroxine (fT4) and increase in plasma thyroid stimulating hormone (TSH) over doses of 0,10,100 and 1000µg/kg/day. Thyroids from treated rats, and FRTL-5 cells treated with a range of concentrations up to 25nM showed histological and biochemical signs of disruption, and evidence for generation of reactive oxygen species (Xu et al, 2022). These compounds have been shown to generate reactive oxygen species or oxidative stress in vivo (Amjad et al, 2020; Mondal and Bandyopadhyay, 2023; Unsal et al, 2020; Wang et al, 2023; Yang et al, 2020).

The bulk of the empirical evidence described above supports the view that administration of high doses of certain xenobiotics (e.g. bisphenol A, CCL4, DEHP, lambda-cyhalothrin) in vivo results in oxidative stress in the thyroid, frequently with clear histological evidence of structural disruption, including loss of colloidal material, and hypothyroidism; namely reduced plasma concentrations of T3 and T4, and elevated plasma TSH. These compounds are, though, well documented as generators of reactive oxygen species or oxidative stress. Similarly, though, several of the compounds are documented as stimulators of T4 clearance. Furthermore, the relatively long-term duration of the studies, and the high doses used, raises the possibility that any changes in T4 secretion rate are consequences of thyroidal changes brought about by other mechanisms, rather than direct response increased oxidative stress in the thyroid: the thyroidal oxidative stress observed could be a consequence of thyroidal damage, without, itself, necessarily being a cause of reduced T4 synthesis. As a result, the observed changes in plasma T4 concentration could be a consequence of contributions from: <ol> <li>Increased T4 clearance and/or</li> <li>Reduced T4 synthesis and secretion: <ol> <li>Arising from increase in oxidative stress, and/or</li> <li>Arising from other mechanisms that may cause an increase in oxidative stress.</li> </ol> </li> </ol> The balance between these contributions can be expected to differ between compounds. Further evidence regarding timings and the chain of events is necessary to determine cause and effect, and the balance of these contributions for different compounds. In contrast to the empirical evidence presented above, treatment of female wistar rats with 40mg/kg/day bisphenol A for 15 days led to an increase in total T3 of 33% over controls, despite evidence for increase in thyroidal reactive oxygen species (specifically H2O2), reduction in radioactive iodine uptake, and detrimental histological changes in the thyroid gland, including loss of follicular colloid (da Silva et al, 2018).

Moderate Male

Moderate

Adult

Moderate

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b43138032f8> AOPWiki 2023-04-24T04:22:59

2023-05-23T11:58:54

<upstream-id>782c23bc-2272-45d6-8660-ab7da297c85e</upstream-id> <downstream-id>b6353d36-591d-4ff5-baa9-f7e65a1c2f24</downstream-id> Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles across vertebrates. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).

The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).

The biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.

It is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007).  There is more limited evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development.  Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels. Modeling of TH in the rat fetus demonstrates the quantitative relationship between TH synthesis and serum T4 concentrations (Hassan et al., 2017, 2020; Handa et al., 2021). Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and in vivo treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Haselman et al., 2020; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008; Tietge et al., 2010). Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole. Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005).  There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages in mammals. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012). In Xenopus, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020).    In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal THs transferred to the eggs. Embryonic TH synthesis is activated later during embryo-larval development. (See Domain of applicability) Dose-response Evidence: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data is available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). This data was used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013). In Xenopus, dose-responses were demonstrated in both thyroidal T4 and circulating T4 following exposure to three TPO inhibitors (Haselman et al., 2020).

There are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied. For example, Kim et al. (2015) investigated the adverse effects of Triphenyl phosphate (TPP), a substance that disrupts the thyroid system. Therefore, Rat pituitary (GH3) and thyroid follicular cell lines (FRTL-5) were studied. In the GH3 cells, TPP led to an upregulation of the expression of important thyroid genes (tsh, tr alpha and tr beta) while T3, a positive control, downregulated the expression of these genes. In FRTL-5 cells, the expression of nis and tpo genes was significantly upregulated, suggesting that TPP stimulates TH synthesis in the thyroid gland. In zebrafish larvae at the age of 7 days post-fertilisation (dpf), TPP exposure resulted in a significant increase in T3 and T4 concentrations and the expression of genes involved in thyroid hormone synthesis. Exposure to TPP also significantly regulated the expression of genes involved in the metabolism (dio1), transport (ttr) and excretion (ugt1ab) of THs. The down-regulation of the crh and tsh genes in the zebrafish larvae suggests the activation of a central regulatory feedback mechanism that is triggered by the increased T3 levels in vivo. Taken together, these observations indicate that TPP increases TH concentrations in early life stages of zebrafish by disrupting central regulatory and hormone synthesis pathways.

During Xenopus metamorphosis, circulating T4 steadily increases to peak levels at metamorphic climax. Therefore, during Xenopus metamorphosis, this KER is operable at an increased rate as compared to a system that is maintaining steady circulating T4 levels through homeostatic control. In this case, developmental status is a modulating factor for the rates and trajectories of these KEs.

In rats, Hassan et al. (2020) demonstrated in vitro: ex vivo correlations of TPO inhibition using PTU and MMI and constructed a quantitative model relating level of TPO inhibition with changes in circulating T4 levels. They determined that 30% inhibition of TPO was sufficient to decrease circulating T4 levels by 20%. This is further supported by studies of Hassan et al. (2017) and Handa et al. (2021) In Xenopus, Haselman et al. (2020) collected temporal and dose-response data for both thyroidal and circulating T4 which showed strong qualitative concordance of the response-response relationship. A quantitative relationship exists there in, but is yet to be demonstrated mathematically in this species.

Fisher et al. (2013) published a quantitative biologically-based dose-response model for iodine deficiency in the rat. This model provides quantitative relationships for thyroidal T4 synthesis (iodine organification) and predictions of serum T4 concentrations in developing rats. There are other computational models that include thyroid hormone synthesis. Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO.  This model was recently adapted for rats(Leonard et al., 2016) and Hassan et al (2017) have extended it to include the pregnant rat dam in response to TPO inhibition induced by PTU. While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or TH synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO, but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme. Further empirical support for the response-response relationship has been demonstrated in the amphibian model, Xenopus laevis, exposed to TPO inhibitors during pro-metamorphosis (Haselman et al., 2020) wherein temporal profiles were measured for both thyroidal and circulating T4.

Given that the thyroid gland contains follicular lumen space filled with stored thyroglobulin/T4, complete inhibition of thyroid hormone synthesis at a given point in time will not result in an instantaneous decrease in circulating T4. The system will be capable of maintaining sufficient circulating T4 levels until the gland stores are depleted. The time it takes to deplete stored hormone will greatly depend on species, developmental status and numerous other factors. In Xenopus, Haselman et al. (2020) demonstrated an approximately 5 day difference between a significant decrease in thyroidal T4 preceding a significant decrease in circulating T4 while exposed to a potent TPO inhibitor (MMI) continuously during pro-metamorphosis.

This KER is entirely influenced by the feedback loop between circulating T4 originating from the thyroid gland and circulating TSH originating from the pituitary. Intermediate biochemical processes exist within the hypothalamus to affirm feedback and coordinately release TSH from the pituitary. However, quantitative representations of these feedback processes are limited to models discussed previously. In Xenopus, circulating levels of T4 increase through pro-metamorphosis indicating a "release" of feedback to allow circulating levels of T4 to increase and drive metamorphic changes (Sternberg et al., 2011). This provides evidence that homeostatic control of feedback can be developmentally dependent, and likely species dependent.

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b4313962680> AOPWiki 2016-11-29T18:41:33

2022-10-10T08:56:38

<upstream-id>b6353d36-591d-4ff5-baa9-f7e65a1c2f24</upstream-id> <downstream-id>a0efb4fc-db66-4c4f-a45d-9df420a299bc</downstream-id> Hypothyroidism in rats and humans leads to an increase in insulin resistance, as determined from in vivo or ex vivo measurements.

Step 1: Identify primary evidence for clinical overt hypothyroidism leading to increase in IR In order to focus on T4 changes, rather than widespread changes to the hypothalamus-pituitary axis that can be involved in sub-clinical hypothyroidism, a search was made to identify clinical studies messing insulin resistance in the presence of T4 reduction below normal levels (overt hypothyroidism).  The following Pubmed search was performed on 18/04/2023, returning 3 relevant references, spanning the period 2012 - 2021 (Prats-Puig et al; 2012, Nada, 2013; Stepanek et al, 2021).

Impact of clinical overt hypothyroidism on insulin resistance <ol> <li> In a cross-sectional study of 234 euthyroid prepubertal children (113 boys, 121 girls) in Spain, HOMA-IR was compared to serum free T4 (Prats-Puig et al, 2012): <ol> <li>For boys, there was no significant difference.</li> <li>For girls, HOMA-IR increased 30% from the highest to the lowest tertile of serum free T4.</li> </ol> </li> <li>In two groups of healthy S.W. Asian females, 27 with overt hypothyroidism and 15 euthyroid, matched for age and BMI, there was no significant difference between the groups for fasting plasma glucose, insulin or HOMA-IR. Six months after starting thyroxine therapy to return the hypothyroid individuals to the euthyroid state, there was no significant change in fasting glucose or HOMA-IR, although fasting insulin had increased significantly above the level measured originally in the euthyroid cohort. However, the relevant parameters in the euthyroid group were not measured, so no longitudinal comparison was possible.</li> <li>In a study of 1425 middle-aged individuals (317 male, 1108 female) in Poland, divided into 3 groups (euthyroid (EU), subclinical hypothyroidism (SH) and overt hypothyroidism(OH)), HOMA-IR, fasting insulin and two-hour levels of oral glucose tolerance test (OGTT) showed steady, yet insignificant increases from the EU, through SH to OH states (Stepanek et al, 2021). Free T4 showed a positive, but not signicant, correlation with HOMA-IR. The ratio of free T3/free T4 was strongly and sigificantly correlated with HOMA-IR. This indicates that if free T4 decreases, then HOMA-IR will increase if T3 simultaneously decreases to a proportionally lesser extent than T4.</li> </ol>   Evidence gleaned from in vivo and ex vivo rodent studies In vivo and ex vivo experiments in the rat indicate an increase in insulin resistance in hypothyroidism. In young (50-60g) and old (160g) male Wistar rats made hypothyroid by administration of propylthiouracil for 4-5 weeks, leading to dramatic reduction in plasma triiodothyronine (T3) concentration, plasma glucose concentration was increased (approximately 10%) in the presence of an approximately 50% (but not significant) insulin concentration increase, compared to euthyroid animals (Dimitriadis et al, 1989). In a soleus muscle preparation from hypothyroid animals, total glucose disposition was decreased compared to a preparation from euthyroid animals: <ul> <li>rates of lactate formation and glucose oxidation were decreased at all insulin concentrations tested, from physiological to supraphysiological;</li> <li>rate of glycogen synthesis was decreased at supraphysiological insulin concentrations.</li> </ul>

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>MF Specification</th> <th>Effect(s) on the KER</th> <th>Reference(s)</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b43139b5268> AOPWiki 2023-04-03T12:17:50

2023-04-20T08:15:52

Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine

Succinate dehydrogenase inhibition leading to increased insulin resistance

Evgeniia Kazymova

<div> Prakash Patel PhD; Cyprotex Discovery Ltd., An Evotec Company, Alderley Park, Cheshire, SK10 4TG, UK. Simon Thomas PhD; Cyprotex Discovery Ltd., An Evotec Company, Alderley Park, Cheshire, SK10 4TG, UK.   </div>

BY-SA

2.0

Many environmental contaminants are known or suspected to generate adverse effects through multiple mechanisms, within multiple cell types, and potentially acting across multiple biological systems, thus rendering elucidation of cause and effect highly challenging. The purpose of creating this AOP is to collate evidence for a plausible pathway by which inhibition of a mitochondrial enzyme, succinate dehydrogenase, could, via disruption of the hypothalamus-pituitary-thydroid axis, contribute to the development of a clinically significant metabolic disruption, namely an increase in insulin resistance (IR). Key to identifying a metabolic effect mediated by thyroid disruption from a direct xenobiotic impact on metabolism is the existence of evidence that qualitatively or quantitatively differentiates between potential impacts via the two routes. Clinical data support the hypothesis that exposure to DEHP - as determined by renal excretion of its major metabolites - has components of both thyroid-mediated (via reduction in circulating free thyroxine concentration) and direct impacts on increase in IR. In vitro data support the hypotheses that (i) inhibition of succinate dehydrogenase can lead to oxidative stress, and (ii) increasing oxidative stress in the thyroid follicular cells can lead to reduction in thyroid hormone secretion. Thus, a plausible link can be established between thyroidal succinate dehydrogenase inhibition and increase in IR. It must be borne in mind that additional molecular initiating events (MIEs) and alternative pathways to increase in IR are plausible for many xenobiotics.   This AOP was created in order to collate information, and attempt to drive understanding, around several key issues concerning xenobiotic driven disruption of energy metabolism, and the subsequent development of related pathologies. Specifically, the focus is on the nature and extent of the component of this disruption that can potentially be mediated via alteration of thyroid gland function, and consequent changes in circulating thyroid hormone concentrations (Huang et al, 2021). Amongst classes of xenobiotics, fragmentary evidence indicates that this is one possible route - amongst many - by which toxicity of phthalate esters could manifest itself. This evidence includes the observations that : <ol> <li>Phthalate esters can increase reactive oxygen species (ROS) concentrations in multiple in vitro systems and in vivo (see, for example, Mondal and Bandypoadhayay, 2023).</li> <li>Phthalate esters can decrease concentrations of thyroid hormones in vivo (Kim et al, 2019).</li> <li>Phthalate ester exposure has been correlated to pathologies of energy metabolism, such as insulin resistance (Shoshtari-Yeganeh et al, 2019; Gao et al, 2021).</li> <li>Decrease in circulating thyroid hormones has been linked to the development of insulin resistance (Brenta, 2011).</li> </ol> Given the role of thyroid hormones in regulating energy metabolism, including glucose and lipids, the possibility exists for phthalate-induced disruptions in energy metabolism and contribution to subsequent pathologies to be mediated at least, in part, through their impacts on thyroid function. This AOP was developed as part of the ScreenED project, which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 825745.

<h1>Identification of molecular initiating event</h1> A significant body of data indicates that: <ol> <li>Many xenobiotics generate ROS in vitro and/or in vivo (e.g. Amjad et al, 2020; Mondal and Bandypoadhayay, 2023; Unsal et al, 2020; Wang et al, 2023; Yang et al, 2020).</li> <li>ROS generation resulting in oxidative stress can be a contributing factor - potentially the most important factor for some xenobiotics - in the induction of toxic, pathological outcomes (e.g. Amjad et al, 2020; Mondal and Bandypoadhayay, 2023; Unsal et al, 2020; Wang et al, 2023; Yang et al, 2020).</li> <li>Multiple mechanisms of excess ROS generation are known, including mechanisms specific to particular cell types, and mechanisms specific to particular xenobiotics or xenobiotic classes (see ArulJothi et al, 2023, for review)</li> <li>The main source of ROS generation under normal physiological conditions in most cell types is the electron transport chain of the mitochondria (Murphy, 2009).</li> </ol> Consequently, of the multiple potential routes of ROS generation, a mechanism involving increased mitochondrial generation is a strong candidate for a potential MIA. <h1>Identification of adverse outcome</h1> Xenobiotic-induced metabolic disfunction can manifest itself in multiple biochemical and physiolological outcomes. These can be of clinical significance in themselves, of significance when taken in conjunction with other measures, and/or indicators of potential progression to further endpoints that may be of clinical significance. Insulin resistance (IR), a measure of the extent to which cells do not respond normally to insulin, is a component of metabolic syndrome, which, in turn, predisposes to type 2 diabetes and cardiovascular disease. Given this central position in the development of metabolic pathologies, and that it can be determined by simple blood measurements, it represents a quantifiable endpoint of clinical relevance, suitable for identification as a significant adverse outcome in and of itself.

There are currently no examples of IR being used in a regulatory role.

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The first iteration of the AOP has been developed in order to outline a plausible route by which stressors that induce oxidative stress could contribute to the global burden of metabolic pathologies acting, specifically, via reduction in thyroid hormone synthesis, and corresponding reduction in circulating thyroxine levels. It was also considered important to identify a particular mechanism that can induce oxidative stress. For phthalate esters, particularly DEHP, there is documented evidence for the chain of events leading from succinate dehydrogenase inhibition, through an increase in thyroid gland oxidative stress and reduction in circulating thyroxine levels, to an increase in insulin resistance (Huang et al, 2021). In vivo evidence for this AOP supports its applicability to adult humans (Huang et al, 2021).

<table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <caption>Essentiality of Key Events</caption> <thead> <tr> <th scope="row">Key Event</th> <th scope="col">Defining question</th> <th scope="col">High (strong)</th> <th scope="col">Moderate</th> <th scope="col">Low (weak)</th> </tr> </thead> <tbody> <tr> <th scope="row"> </th> <td>Are downstream KEs and/or the AO prevented if an  upstream KE is blocked?</td> <td>Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs</td> <td>Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE</td> <td>No or contradictory experimental evidence of the essentiality of any of the KEs</td> </tr> <tr> <th scope="row" style="text-align:left">KE 2118 (MIE): Succinate dehydrogenase, inhibited</th> <td> </td> <td> </td> <td> </td> <td>No direct evidence identified</td> </tr> <tr> <th scope="row" style="text-align:left">KE 2120: Superoxide generation, increased</th> <td> </td> <td> </td> <td> </td> <td>No direct evidence identified</td> </tr> <tr> <th scope="row" style="text-align:left">KE 1969: Increase, Oxidative Stress</th> <td> </td> <td> </td> <td> </td> <td> </td> </tr> <tr> <th scope="row" style="text-align:left">KE 277: Thyroid hormone synthesis, Decreased</th> <td> </td> <td> </td> <td>Pharmacological inhibition of thyroid synthesis has been observed to lead to increase in insulin resistance in animals.</td> <td> </td> </tr> <tr> <th scope="row" style="text-align:left">KE 281: Thyroxine (T4) in serum, Decreased</th> <td> </td> <td> </td> <td>Reduction in T4 concentration contributing to an increase in insulin resistance in humans has been inferred using mediation analysis (Huang et al, 2021).</td> <td> </td> </tr> </tbody> </table>

<table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <caption>Plausibility of Key Event Relationships</caption> <thead> <tr> <th scope="row">Key Event Relationship</th> <th scope="col">Defining question</th> <th scope="col">High (strong)</th> <th scope="col">Moderate</th> <th scope="col">Low (weak)</th> </tr> </thead> <tbody> <tr> <th scope="row"> </th> <td>Is there a mechanistic (i.e., structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?</td> <td>Extensive understanding based on extensive previous documentation and broad acceptance -Established mechanistic basis</td> <td>The KER is plausible based on analogy to accepted biological relationships but scientific understanding is not completely established.</td> <td>There is empirical support for a statistical association between KEs (See 3.), but the structural or functional relationship between them is not understood.</td> </tr> <tr> <th scope="row" style="text-align:left">SDH, inhibited leads to superoxide generation, increased</th> <td> </td> <td>Inhibition of mitochondrial succinate dehydrogenase has been demonstrated to lead to an increase in mitochondrial superoxide generation in in vitro studies</td> <td> </td> <td> </td> </tr> <tr> <th scope="row" style="text-align:left">Superoxide generation, increased leads to Increase, Oxidative Stress</th> <td> </td> <td>Activation of superoxide generation, and superoxide dismutase overexpression have been shown to lead, respectively, to increase and decrease of markers of oxidative stress within cells.</td> <td> </td> <td> </td> </tr> <tr> <th scope="row" style="text-align:left">Increase, Oxidative Stress leads to TH synthesis, Decreased</th> <td> </td> <td> </td> <td>Increased oxidative stress leads to adverse biochemical and histological outcomes in thyroid cells in vitro and in vivo, including reduced follicular colloidal amounts</td> <td> </td> </tr> <tr> <th scope="row" style="text-align:left">TH synthesis, Decreased leads to T4 in serum, Decreased</th> <td> </td> <td>Inhibition of T4 synthesis in vivo is expected to  lead to reduction in circulating T4 concentration, though mediated by the counterbalancing effect of negative feedback in the</td> <td> </td> <td> </td> </tr> <tr> <th scope="row" style="text-align:left">T4 in serum, Decreased leads to Insulin resistance, increased</th> <td> </td> <td>Decreased T4 serum concentration has been demonstrated to result in increase in insulin resistance in rodent studies and in humans</td> <td> </td> <td> </td> </tr> </tbody> </table>

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

Quantitative understanding of the AOP is currently low.

Not Specified

Amjad, S. et al (2020), "Role of antioxidants in alleviating bisphenol A toxicity", Biomolecules, Vol 10, 1105. ArulJothi, K.N. et al (2023), "Implications of reactive oxygen species in lung cancer and exploiting it for therapeutic interventions", Medical Oncology, Vol 40, 43. Brenta, G. (2011), "Why can insulin resistance be a natural consequence of thyroid dysfunction?", Journal of Thyroid Research, 152850. Gao, H. et al (2021), "Association between phthalate exposure and insulin resistance: a systemic review and meta-analysis update", Environmental Science and Pollution Research International", Vol 28, pp 55967-55980. Huang, H-B et al (2021), "Mediation effects of thyroid function in the associations between phthalate exposure and glucose metabolism in adults", Environmental Pollution Vol 278, 116799. Kim, M.J. et al (2019), "Association between Diethylhexyl phthalate exposure and thyroid function: a meta-analysis", Thyroid, Vol 29, pp 183-192, with correction in Vol 29, 752. Mondal, S. and Bandyopadhyay, A. (2023), "From oxidative imbalance to compromised standard sperm parameters: toxicological aspects of phthalate esters on spermatozoa", Environmental Toxicology and Pharmacology, Vol 98, 104085. Murphy, M.P. (2009), "How mitochondria produce reactive oxygen species", Biochemical Journal, Vol 417, pp 1-13. Shoshtari-Yeganeh, B, et al (2019) "Systematic review and meta-analysis on the association between phthalates exposure and insulin resistance", Environmental Science and Pollution Research International", Vol 26, pp 9435-9442. Unsal, V. et al (2020), "Toxicity of carbon tetrachloride, free radicals and role of antioxidants", Research in Environmental Health, Vol 36, pp279-295. Wang, Q.-Y. et al (2023), "2,3',4,4',5-Pentachlorophenol induces mitochondria-dependent apoptosis mediated by AhR/Cyp1a1 in mouse germ cells", Journal of Hazard Materials, Vol 445, 130457. Yang, C. et al (2020), "Mediation of oxidative stress toxicity induced by pyrethroid pesticides in fish", Comp Biochem Physiol C Toxicol Pharmacol., Vol 234, 108758. AOPWiki 2022-06-30T09:47:52

2023-09-25T16:27:12