CYP2E1 Activation

60-35-5

DLFVBJFMPXGRIB-UHFFFAOYSA-N

Acetamide

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

DTXSID7020005

103-90-2

RZVAJINKPMORJF-UHFFFAOYSA-N

Acetaminophen

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

DTXSID2020006

968-81-0

VGZSUPCWNCWDAN-UHFFFAOYSA-N

Acetohexamide

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

DTXSID7020007

13838-16-9

JPGQOUSTVILISH-UHFFFAOYNA-N

JPGQOUSTVILISH-UHFFFAOYSA-N

Enflurane

Ethane, 2-chloro-1-(difluoromethoxy)-1,1,2-trifluoro- (.+-.)-Enflurane 2-Chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane 2-Chloro-1,1,2-trifluoro-1-difluoromethoxyethane 2-Chloro-1,1,2-trifluoroethyl difluoromethyl ether Alyrane Enfluran enflurano Ether, 2-chloro-1,1,2-trifluoroethyl difluoromethyl Ethrane Methylflurether NSC 115944

DTXSID1020562

151-67-7

BCQZXOMGPXTTIC-UHFFFAOYNA-N

BCQZXOMGPXTTIC-UHFFFAOYSA-N

Halothane

Ethane, 2-bromo-2-chloro-1,1,1-trifluoro- (.+-.)-Halothane 1,1,1-Trifluoro-2-bromo-2-chloroethane 1,1,1-Trifluoro-2-chloro-2-bromoethane 1-Bromo-1-chloro-2,2,2-trifluoroethane 2,2,2-Trifluoro-1-chloro-1-bromoethane 2-Bromo-2-chloro-1,1,1-trifluoroethane 2-Chloro-2-bromo-1,1,1-trifluoroethane Alotano Anestan Fluktan Fluothane Freon 123B1 Ftorotan halotano Halothan Narcotan Narcotane Narkotan NSC 143490 Rhodialothan UN 1610

DTXSID4025371

26675-46-7

PIWKPBJCKXDKJR-UHFFFAOYNA-N

PIWKPBJCKXDKJR-UHFFFAOYSA-N

Isoflurane

Ethane, 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro- (.+-.)-Isoflurane 1-Chloro-2,2,2-trifluoroethyl difluoromethyl ether 2-Chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane Aerrane Difluoromethyl 1-chloro-2,2,2-trifluoroethyl ether Ether, 1-chloro-2,2,2-trifluoroethyl difluoromethyl HCFE 235da2 Isofluran isoflurano Isoforine

DTXSID3020752

76-38-0

RFKMCNOHBTXSMU-UHFFFAOYSA-N

Methoxyflurane

Ethane, 2,2-dichloro-1,1-difluoro-1-methoxy- (2, 2-Dichloro-1, 1-difluoroethyl) methyl ether 1,1-Dichloro-2,2-difluoro-2-methoxyethane 2,2-Dichloro-1,1-difluoro-1-methoxyethane 2,2-Dichloro-1,1-difluoroethyl methyl ether Analgizer Anecotan Ether, 2,2-dichloro-1,1-difluoroethyl methyl Inhalan Methoflurane Methoxane Methoxyfluran Methyl 1,1-difluoro-2,2-dichloroethyl ether Metofane Metoxfluran Metoxifluran metoxiflurano NSC 110432 Penthrane Pentran Pentrane

DTXSID7025556

28523-86-6

DFEYYRMXOJXZRJ-UHFFFAOYSA-N

Sevoflurane

Propane, 1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)- 1,1,1,3,3,3-Hexafluoro-2-(fluoromethoxy)propane 347mmzEβγ Ether, fluoromethyl 2,2,2-trifluoro-1-(trifluoromethyl)ethyl Ether, fluoromethyl 2,2,2-trifluoro-1-(trifluoromethyl)ethyl- Fluoromethyl 1,1,1,3,3,3-hexafluoro-2-propyl ether Fluoromethyl 2,2,2-trifluoro-1-(trifluoromethyl)ethyl ether Sevofluran Sevofrane Sevorane

DTXSID8046614

95-25-0

TZFWDZFKRBELIQ-UHFFFAOYSA-N

Chlorzoxazone

2(3H)-Benzoxazolone, 5-chloro- 2(3H)-Benzoxazoline, 5-chloro- 2-Benzoxazolinone, 5-chloro- 2-Hydroxy-5-chlorobenzoxazole 5-Chloro-1,3-benzoxazol-2(3H)-one 5-Chloro-2(3H)-benzoxazolone 5-Chloro-2-benzoxazolinone 5-Chloro-2-benzoxazolol 5-Chloro-2-benzoxazolone 5-Chloro-2-hydroxybenzoxazole 5-Chloro-2-oxo-3H-benzoxazole 5-Chloro-3H-benzoxazol-2-one 5-Chlorobenzoxazolidone 5-Chlorobenzoxazolone Biomioran Chloroxazone Chlorzoxazon clorzoxazona Escoflex Miotran Myoflexin Myoflexine Neoflex NSC 26189 Paraflex Parafon Forte DSC Pathorysin Solaxin

DTXSID9022813

12137-20-1

OGIDPMRJRNCKJF-UHFFFAOYSA-N

Titanium oxide (TiO)

oxido de titanio Oxyde de titane Titan Black 12S Titanium Black 12S titanium monoxide Titanoxid

DTXSID7065253

54-85-3

QRXWMOHMRWLFEY-UHFFFAOYSA-N

Isoniazid

INH 4-Pyridinecarboxylic acid, hydrazide [(4-Pyridinylcarbonyl)oxy]hydrazine 4-(Hydrazinocarbonyl)pyridine 4-Pyridinecarbonylhydrazine 4-Pyridinecarboxylic acid hydrazide 4-Pyridinecarboxylic acid hydrazine 4-Pyridinecarboxylic hydrazide 4-Pyridylcarbonylhydrazide Antimicina Armazid Armazide Atcotibine Cotinazin Dianicotyl Dinacrin Ditubin Ertuban Eutizon Hidranizil Hidrasonil Hycozid Hydrazid Isidrina Isobicina Isocotin Isoniazid SA isoniazida Isoniazide Isonicid Isonico Isonicotan ISONICOTINIC ACID HYDRAZIDE Isonicotinic hydrazide Isonicotinohydrazide Isonicotinoyl hydrazide Isonicotinoylhydrazine ISONICOTINSAEURE-HYDRAZID Isonidrin Isonilex Isonindon Isonizide Isotebezid Isozide Laniazid Mayambutol Mybasan Neoteben Neumandin Niadrin Nicetal Nicizina Niconyl Nicotibina Nicotisan Nicozide Nidaton Nidrazid Nikozid Nitadon NSC 9659 Nydrazid Nyscozid Pelazid Preparation 6424 Pycazide Pyricidin PYRIDINE, 4-CARBOXYLIC ACID HYDRAZIDE Pyridine-4-carbohydrazide Pyridine-4-carboxylate hydrazide Pyrizidin Raumanon Retozide Rimicid Rimifon Rimiphone Rimitsid Robisellin RU-EF-Tb Sauterazid T.B. Razide Tebecid Teebaconin Tekazin Tibinide Tibizide Tisiodrazida Tubazid Tubazide Tubicon Tubilysin Tubomel Unicozyde Vazadrine Vederon Zidafimia Zinadon Zonazide

DTXSID8020755

64-17-5

LFQSCWFLJHTTHZ-UHFFFAOYSA-N

Ethanol

Ethyl alcohol AETHANOL Alcare Hand Degermer Alcohol Alcohol anhydrous Algrain Anhydrol Anhydrol PM 4085 Denatured alcohol Denatured ethanol Denatured ethyl alcohol Desinfektol EL Duplicating Fluid 100C.NPA Esumiru WK 88 Ethicap Ethyl hydrate Ethyl hydroxide Hinetoless Infinity Pure Jaysol S Methylcarbinol Molasses alcohol NSC 85228 Potato alcohol Sekundasprit Sterillium Rub SY Fresh M Synasol Tecsol C UN 1170 UN1170 Vinic alcohol EtOH

DTXSID9020584

79-06-1

HRPVXLWXLXDGHG-UHFFFAOYSA-N

Acrylamide

2-Propenamide 2-Propene amide acrilamida Acrylamid Acrylamide monomer Acrylic acid amide Acrylic amide Bio-Acrylamide 50 Ethylenecarboxamide NSC 7785 Propenamide UN 2074 UN3426 Vinyl amide

DTXSID5020027

541-53-7

JIRRNZWTWJGJCT-UHFFFAOYSA-N

2,4-Dithiobiuret

Dicarbonodithioimidic diamide Dithioimidodicarbonic diamide

DTXSID2034968

110-13-4

OJVAMHKKJGICOG-UHFFFAOYSA-N

2,5-Hexanedione

Acetonylacetone 1,2-Diacetylethane 2,5-Diketohexane 2,5-Dioxohexane ACETONYL ACETONE ACETONYLACETON Diacetonyl Hexan-2,5-dion Hexane-2,5-dione hexano-2,5-diona NSC 7621 α,β-Diacetylethane

DTXSID8030138

100-42-5

PPBRXRYQALVLMV-UHFFFAOYSA-N

Styrene

Ethenylbenzene Phenylethylene Vinylbenzene Benzene, ethenyl- Benzene ethenyl Benzene,ethenyl- Benzene,ethenyl.. Cinnamene estireno Fenil etileno NSC 62785 Phenethylene Phenylethene Stypol 040-0165 STYRENE MONOMER Styrole Styrolene Styropol SO UN 2055 VINYL BENZENE, PHENYLETHYLENE Vinylbenzene, Phenylethylene Vinylbenzol

DTXSID2021284

107-13-1

NLHHRLWOUZZQLW-UHFFFAOYSA-N

Acrylonitrile

2-Propenenitrile acrilonitrilo ACRYLNITRIL Acrylon Acrylonitril Carbacryl Cyanoethene Cyanoethylene Fumigrain NSC 6362 PROP-2-ENENITRILE Propenenitrile UN 1093 Vinyl cyanide

DTXSID5020029

107-02-8

HGINCPLSRVDWNT-UHFFFAOYSA-N

Acrolein

2-Propenal 2-Propen-1-al 2-Propen-1-one acrilaldehido Acroleina Acrylaldehyd Acrylaldehyde Acrylic aldehyde Allyl aldehyde Aqualin Magnacide B Magnacide H NSC 8819 Prop-2-en-1-al Propenal UN 1092

DTXSID5020023

51-75-2

HAWPXGHAZFHHAD-UHFFFAOYSA-N

Nitrogen mustard

HN-2 Ethanamine, 2-chloro-N-(2-chloroethyl)-N-methyl- 2,2'-Dichloro-N-methyldiethylamine BIS-(2-CHLORAETHYL)-METHYL-AMIN Bis(2-chloroethyl)methylamine Bis(β-chloroethyl)methylamine Chlorethazine Chlormethin Chlormethine Cloramin clorometina Di(2-chloroethyl)methylamine Diethylamine, 2,2'-dichloro-N-methyl- Methylbis(2-chloroethyl)amine Methylbis(β-chloroethyl)amine Methyldi(2-chloroethyl)amine Methyl-β,β-dichlorodiethylamine Mustargen Mustine Mustine Note N,N-Bis(2-chloroethyl)methylamine N,N-Di(chloroethyl)methylamine Nitrogen mustard [2-Chloro-N-(2-chloroethyl)-N-methylethanamine] N-Methyl-2,2'-dichlorodiethylamine N-Methylbis(2-chloroethyl)amine N-Methylbis(β-chloroethyl)amine NSC 128663 Mechlorethamine

DTXSID2020975

Acetaminophen

2016-11-29T18:42:26

Enflurane

2018-04-05T06:31:22

Halothane

2018-04-05T06:32:13

Isoflurane

2018-04-05T06:32:26

Methoxyflurane

2018-04-05T06:32:50

Sevoflurane

2018-04-05T06:33:06

Chemical:584015 (1-~13~C)Aniline

2018-04-05T06:33:32

Chlorzoxazone

2018-04-05T06:34:56

Titanium oxide (TiO)

2018-04-05T06:35:41

Isoniazid

2018-04-05T06:36:00

Ethanol

2018-04-05T06:38:48

Acrylamide

2017-11-08T11:15:19

2,4-Dithiobiuret

2018-03-26T09:42:51

2,5-Hexanedione

2018-04-05T06:50:57

Styrene

2018-04-05T06:52:41

4-Hydroxy Nonenal

2018-04-05T06:53:21

Acrylonitrile

2018-04-05T06:54:38

Acrolein

2017-08-15T09:55:59

2021-09-28T08:23:20

Nitrogen mustard

2018-04-05T06:55:24

Acetyldehyde

2018-04-05T06:55:41

WCS_9606

common toxicological species human

CYP2E1 Activation

Molecular

CYP2E1 is an enzyme which is part of the cytochrome P450 family. It participates in the metabolism of endogenous, small and hydrophobic compounds using an oxidation reaction. CYP2E1 requires phospholipids, NADPH, and NADPH-cytochrome P450 reductase as a cofactor for catalytic activity in vitro. The level of CYP2E1 in the brain of a rat is significantly low, only 25% of what is found in rat liver cells. Inside a brain cell of a rat the highest level of CYP2E1 is found in the mitochondria and the endoplasmic reticulum. In human brain samples expression of CYP2E1 was found in the amygdala and prefrontal cortex. CYP2E1 is also known to activate toxic related compounds (see stressors). Another important consideration is that CYP2E1 is part of the microsomal ethanol oxidizing system. When CYP2E1 is stimulated at constant rate the concentration of the enzyme in the cell will rise. (Garciá-Suástegui, W. A. et al., 2017; Howard, L. A. et al., 2003; Neafsey, P. et al., 2009; Toselli, F. et al, 2015; Zimatkin, S. M. et al, 2006)

Visualization of CYP2E1 can be done with immunofluorescence in cultured neuron cells. With the use of this technique, an increase or decrease of CYP2E1 can be measured. A rabbit polyclonal antibody for CYP2E1 is used in combination with a FITC-conjugated anti-rabbit antibody. With the FITC tag the CYP2E1 proteins can be made visible using fluorescence. Measurements are done with a confocal laser-scanning microscope. Another way of visualization can be done with the western blot analysis. (Garciá-Suástegui, W. A. et al., 2017; Valencia-Olvera, A. C. et al., 2014)

UBERON:0000451

UBERON prefrontal cortex

Garciá-Suástegui, W. A. et al. The Role of CYP2E1 in the Drug Metabolism or Bioactivation in the Brain. Oxidative Medicine and Cellular Longevity 2017, (2017) Howard, L. A., Miksys, S., Hoffmann, E., Mash, D. & Tyndale, R. F. Brain CYP2E1 is induced by nicotine and ethanol in rat and is higher in smokers and alcoholics. Br. J. Pharmacol. 138, 1376–1386 (2003). Neafsey, P. et al. Genetic polymorphism in CYP2E1: Population distribution of CYP2E1 activity. Journal of Toxicology and Environmental Health - Part B: Critical Reviews 12, 362–388 (2009) Toselli, F. et al. Expression of CYP2E1 and CYP2U1 proteins in amygdala and prefrontal cortex: Influence of alcoholism and smoking. Alcohol. Clin. Exp. Res. 39, 790–797 (2015) Valencia-Olvera, A. C., Morán, J., Camacho-Carranza, R., Prospéro-García, O. & Espinosa-Aguirre, J. J. CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons. Toxicol. Vitr. 28, 1206–1214 (2014) Zimatkin, S. M., Pronko, S. P., Vasiliou, V., Gonzalez, F. J. & Deitrich, R. A. Enzymatic mechanisms of ethanol oxidation in the brain. Alcohol. Clin. Exp. Res. 30, 1500–1505 (2006) AOPWiki 2018-03-26T09:19:36

2018-04-09T11:02:10

Protein Adduct Formation

Molecular

Reactive chemicals or metabolites can interact with proteins present in any cell type which occur at the molecular level. The electrophilic chemicals react with the nucleophilic parts of proteins, forming a covalent bond. When proteins are in their original shape they can function properly, when this is not the case the protein loses its function. These are unspecific proteins which are altered in shape due to the covalent binding of chemicals.

The determination of protein adducts can be done, but since some chemicals induce protein adducts at low abundance it is hard to measure. A new technique is developed to overcome the problem of the low abundance. A drug is labelled with a biotin affinity tag, which than can react with proteins and bind covalently. After incubation with several proteins the drugs attached to the proteins are separated from the rest of the free proteins with the use of the tag. Next the proteins which are altered by the drug can be identified with proteomics. A LC/MS/MS technique is used. A problem which can occur, is that the use of a tag can influence the binding profile of the drug. Another possibility is the use of 2D gel-electrophoresis and tandem mass spectrometry based proteomics. Proteins are purified from cells after incubation with a drug. With 2D gel-electrophoresis the proteins are separated, afterwards the spots of interest are identified with tandem mass spectrometry.

UBERON:0000955

UBERON brain

LoPachin, R. M. & DeCaprio, A. P. Protein adduct formation as a molecular mechanism in neurotoxicity. Toxicological Sciences 86, 214–225 (2005) Gan, J., Zhang, H. & Humphreys, W. G. Drug-Protein Adducts: Chemistry, Mechanisms of Toxicity, and Methods of Characterization. Chemical Research in Toxicology 29, 2040–2057 (2016) AOPWiki 2018-03-26T09:35:55

2018-03-26T09:45:40

Oxidative Stress in Brain

Molecular

Oxidative stress is the imbalance between ROS and defence mechanisms against these ROS. Due to this imbalance the concentration of ROS can rise in the cells. The oxidizing free radicals can cause damage in the cell, at the DNA but also on multiple proteins. Nrf2 is an example of an defence mechanism against ROS.

One way to show oxidative stress in cells is to measure the ROS level. ROS generation can be determined in neuron cells. DHE, a small-molecule ROS probe, can be used for visualizing ROS generation with the use of an epifluorescence microscope. DHE is a direct way of measuring ROS, an indirect way is the measurement of glutathione depletion. The ratio between glutathione and oxidized glutathione can be determined, which shows indirectly whether the ROS level is increased. When oxidized glutathione is present in a higher concentration the ROS level is increased. Finally the expression level of Nrf2 can be determined in cells, with the use of a western blot analysis and antibodies for Nrf2. When the expression level of Nrf2 is much higher than in a control cell it indirectly shows that there is an increase in the concentration of ROS.

Halpin, L. E., Collins, S. A. & Yamamoto, B. K. Neurotoxicity of methamphetamine and 3,4-methylenedioxymethamphetamine. Life Sciences 97, 37–44 (2014). Valencia-Olvera, A. C., Morán, J., Camacho-Carranza, R., Prospéro-García, O. & Espinosa-Aguirre, J. J. CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons. Toxicol. Vitr. 28, 1206–1214 (2014). Nguyen, T., Nioi, P. & Pickett, C. B. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 284, 13291–5 (2009).   AOPWiki 2018-04-04T14:18:29

2018-04-04T14:24:00

Lipid Peroxidation

Molecular

Lipid peroxidation is the direct damage to lipids in the membrane of the cell or the membranes of the organelles inside the cells. Ultimately the membranes will break due to the build-up damage in the lipids This is mainly caused by oxidants which attack lipids specifically, since these contain carbon-carbon double bonds. During lipid peroxidation several lipid radicals are formed in a chain reaction. These reactions can interfere and stimulate each other. Antioxidants, such as vitamin E, can react with lipid peroxy radicals to prevent further damage in the cell. Main products of the lipid peroxidation reaction are lipid hydroperoxides, malondialdehyde and 4-hydroxynonenal.

Proteomics is a tool which can be used for the detection of 4-hydroxynonenal modified proteins. Mitochondrial and cytosolic proteins are subjected to 2D SDS-PAGE and are separated based on the size and pH. With the use of a polyclonal antibody against 4-hydroxynonenal which has an immunofluorescence tag the modified proteins can be detected. The proteins of interest on the SDS-PAGE gel can be cut out and cleaned. Cleaning is necessary since the identification is done by a MALDI-TOF MS analysis.

Ayala, A., Muñoz, M. F. & Argüelles, S. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014, (2014) Andringa, K. K., Udoh, U. S., Landar, A. & Bailey, S. M. Proteomic analysis of 4-hydroxynonenal (4-HNE) modified proteins in liver mitochondria from chronic ethanol-fed rats. Redox Biol. 2, 1038–1047 (2014) Smathers, R. L., Galligan, J. J., Stewart, B. J. & Petersen, D. R. Overview of lipid peroxidation products and hepatic protein modification in alcoholic liver disease. in Chemico-Biological Interactions 192, 107–112 (2011)   AOPWiki 2018-04-04T14:25:07

2018-04-04T14:33:00

Unfolded Protein Response

Unfolded Prortein Response

Molecular

The endoplasmic reticulum is responsible for the synthesis of proteins that are secreted from the cell. The folding of these proteins inside the endoplasmic reticulum is a precise mechanism, however the folding capacity is limited. Building block for the folding of the proteins are chaperones and foldases. Stress in the endoplasmic reticulum starts when the demand for protein folding increases over the protein folding capacity. Another factor is the accumulation of unfolded proteins in the endoplasmic reticulum which can cause stress. The stress leads to the activation of the UPR. Three effectors are released: ATF6, IRE1 and PERK. During the release chaperone BiP (GRP78) is removed from the effectors which maintain them in inactive state. The role of the unfolded protein response is to maintain the protein homeostasis in the endoplasmic reticulum.   Description from EU-ToxRisk: Activation of UPR sensors (IRE1/PERK/ATF6) -> activation of transcription factors (XBP1/ATF4/ATF6f) -> activation of downstream targets (CHOP &Bip)

GRP78 can be used as a biomarker for endoplasmic stress and as discussed before this protein is released from the effector when the unfolded protein reaction occurs. The detection of the GRP78 protein is possible with the use of 2D gel electrophoresis in combination with proteomics. Proteins are extracted from cells, which were incubated with a toxicant, and sampled on the 2D gel. Separation is done based on the protein size and the pH value. Because of the low abundance the GRP78 protein can also be made visible with immunohistochemistry and imaging using an anti-GRP78 antibody. The protein of interest form the 2D gel can be purified and cleaned for the identification with mass spectrometry.   Description from EU-ToxRisk: Activation of the sensors, transcription factors and targets can be measured through several assays (Takayanagi, et al 2013) and fluorescent reporter cell lines (Wink et al., 2014)

Yang, F. & Luo, J. Endoplasmic reticulum stress and ethanol neurotoxicity. Biomolecules 5, 2538–2553 (2015). Guan, M. et al. MDA-9 and GRP78 as potential diagnostic biomarkers for early detection of melanoma metastasis. Tumour Biol. 36, 2973–82 (2015). Foufelle, F. & Fromenty, B. Role of endoplasmic reticulum stress in drug-induced toxicity. Pharmacol. Res. Perspect. 4, e00211 (2016). Takayanagi, S., Fukuda, R., Takeuchi, Y., Tsukada, S., & Yoshida, K. (2013). Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress. Cell Stress and Chaperones, 18(1), 11–23. <a href="https://doi.org/10.1007/s12192-012-0351-5">https://doi.org/10.1007/s12192-012-0351-5</a> Wink, S., Hiemstra, S., Huppelschoten, S., Danen, E., Niemeijer, M., Hendriks, G., … Van De Water, B. (2014). Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment. Chemical Research in Toxicology, 27(3), 338–355. https://doi.org/10.1021/tx4004038 AOPWiki 2018-04-04T14:25:43

2019-03-07T09:47:23

General Apoptosis

Cellular

Apoptosis is the programmed cell death in general. This process is well regulated with a sequence of events before cell fragmentation occurs. Changes in the nucleus of a neuron cell are the first step in apoptosis. Before that, other factors such as stress, inflammation, cell damage can induce expression or activation of signal proteins which will activate the pathway for apoptosis. Examples of proteins which are involved in apoptosis are the proteins p53, Bcl-2, JNK, and several caspases. When the first step is taken in the apoptosis process the neuron cell will end in membrane-bounded apoptotic bodies. These bodies are cleared by macrophages or other cells where the degradation process starts within heteorphagosomes.

There are several possibilities to measure and detect apoptosis, some examples are the detection of LDH and MTT substances which are released from cells which commit suicide. An older but effective technique it the annexin V – affinity assay. The principle of this assay is the high affinity binding between annexin V and phosphatidylserine. In a vital cell there is a membrane lipid asymmetry where phosphatidylserine molecules are facing the cytosol. During apoptosis the membrane lipid asymmetry is lost, and the phosphatidylserine molecules are expressed in the outer membrane. When annexin-V is present in combination with Ca2+ it binds with high affinity to phosphatidylserine. With a hapten label at the annexin-V this process can be detected. Other techniques are the detection of cleaved caspase-3, which could be done with a western blot or enzyme-linked immunosorbent assays. Cytochrome c is also a protein which is released in an early stage of apoptosis. Detection of cytochrome c can be done with metal nanoclusters which have a fluorescent probe.

Shtilbans, V., Wu, M. & Burstein, D. E. Evaluation of apoptosis in cytologic specimens. Diagnostic Cytopathology 38, 685–697 (2010). Wu, J., Sun, J. & Xue, Y. Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells. Toxicol. Lett. 199, 269–276 (2010). Redza-Dutordoir, M. & Averill-Bates, D. A. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim. Biophys. Acta - Mol. Cell Res. 1863, 2977–2992 (2016). Lossi, L., Castagna, C. & Merighi, A. Neuronal cell death: An overview of its different forms in central and peripheral neurons. in Neuronal Cell Death: Methods and Protocols 1–18 (2014). doi:10.1007/978-1-4939-2152-2_1 Van Engeland, M., Nieland, L. J. W., Ramaekers, F. C. S., Schutte, B. & Reutelingsperger, C. P. M. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1–9 (1998). Shamsipur, M., Molaabasi, F., Hosseinkhani, S. & Rahmati, F. Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes. Anal. Chem. 88, 2188–2197 (2016). AOPWiki 2018-04-04T14:30:27

2018-04-04T14:51:14

Neurodegeneration

Organ

Neurodegeneration is the loss of neuron cells in the brain and spinal cord. The loss of neurons can either be functional loss, also called ataxia, or sensory dysfunction, known as dementia. The brain is organized in different parts which all have their own function in an individual. So which affect the neurodegeneration has on an individual it depends on where neuron loss occurs.

A biomarker is needed since a common way to detect neurodegeneration is with the use of a PET scan. Although this technique works, another in vitro method needed to predict whether a chemical can induce neurodegeneration before it damages a whole brain. A possible biomarker of neurodegeneration can be found in the cerebrospinal fluid. These biomarkers are the spectrin breakdown products, specifically SBDP-145. There are two possible ways of measuring SBDP-145. First quantification can be done with an ELISA technique, from a sample taken out of the cerebrospinal fluid. Another way is to label SBDP-145 with the use of immunohistochemistry, so that the expression and release of SBDP-145 can be made visible.

Uttara, B., Singh, A. V, Zamboni, P. & Mahajan, R. T. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr. Neuropharmacol. 7, 65–74 (2009). Pritt, M. L. et al. Initial biological qualification of SBDP-145 as a biomarker of compound-induced neurodegeneration in the rat. Toxicol. Sci. 141, 398–408 (2014). AOPWiki 2018-04-04T14:30:58

2018-04-04T14:53:32

<upstream-id>b8b9160f-0fe9-4a96-b41e-327e61c1f561</upstream-id> <downstream-id>132dff3c-afb4-4c71-a841-a9e99c720064</downstream-id> CYP2E1 is part of the cytochrome P450 family and can participate in the metabolism of endogenous, small and hydrophobic compounds using an oxidation reaction.  When CYP2E1 is activated it can induce ROS formation. Activation of CYP2E1 will also lead to an increased expression of the enzyme itself, which will ultimately increase the formation of ROS. CYP2E1 is expressed at various parts in the human brain, such as cortex, cerebellum, hippocampus, thalamus and stratum. Since the level of defence mechanism in the brain against ROS is lower than in other parts in the body oxidative stress is reached faster.

The link between CYP2E1 activation and the formation of ROS, which ultimately leads to oxidative stress, was already made in the 90s. Hydroxyethyl free radicals where found in rat livers after the stimulation of CYP2E1, which eventually leads to liver damage.  As already mentioned in the KE description, oxidative stress is defined as the moment when there is an imbalance between the ROS level and the defence mechanisms which leads to damage in the cell. Research done by Haorah et al. showed that CYP2E1 indeed produces ROS. ROS levels where measured in two different situations, neuron cells where induced with ethanol (inducer of CYP2E1) or neuron cells where induced with ethanol in combination with an inhibitor for CYP2E1. ROS levels in the neuron cells decreased significantly with the inhibitor for CYP2E1 when compared with the situation without the inhibitor for CYP2E1. Three other studies, using different cell types, showed that CYP2E1 KO mice resulted in an increased level of TBARS (marker for lipid peroxidation which is induced by ROS).  Also in two of the three studies a higher level of GSH was detected in CYP2E1 KO mice, indicating a lower level of ROS since GSH is used as a defence mechanism against ROS. Furthermore recent research showed that CYP2E1 induction in granule neurons indeed results in ROS formation, but also that the inducement of CYP2E1 increased the expression of CYP2E1 itself. This was also shown in other studies, with the use of immunofluorescence detection techniques. Since activation of CYP2E1 also leads to a higher expression more ROS will be produced. This is also shown in the difference of CYP2E1 expression in alcoholics and non-drinkers, where the expression of CYP2E1 is far higher in alcoholic liver cells. Finally, oxidative stress is reached earlier in neuron cells because of the higher level of oxygen and the lower permeability of the blood vessels.

The link between CYP2E1 activation oxidative stress is biological plausible.

Many studies are performed with ethanol, which is a well-known inducer of CYP2E1. But ethanol can also induce ROS formation by interfering in other biological pathways or inducing endoplasmic reticulum stress, which eventually can lead to neurotoxicity and neurodegeneration. On the other hand direct evidence is available with the studies described above that CYP2E1 induces ROS. Important studies performed are the WT/KO/KI mice and the detection of further CYP2E1 expression when CYP2E1 is activated.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea288540> AOPWiki 2018-04-04T15:02:06

2018-04-05T03:40:35

<upstream-id>132dff3c-afb4-4c71-a841-a9e99c720064</upstream-id> <downstream-id>4cbe7b00-a84e-412e-9a54-64403998fef7</downstream-id> Lipid peroxidation is a following event after oxidative stress. During oxidative stress the level of ROS is rising, which increases the concentration of free radical species. These highly unstable free radicals can easily react with macromolecules such as lipids. The brain has a high level of PUFAs and neuronal cells are known to be relatively unable to neutralize free radicals. Together with the knowledge that free radicals mainly attack PUFAs make neuron cells vulnerable for lipid peroxidation. The reaction between free radicals and PUFAs leads to the formation of highly reactive electrophilic aldehydes, such as MDA and HNE.  Lipid peroxidation can be described by 5 steps. The initiation of the free radical, production of peroxyl radical, self-perpetuating chain reaction (leading to several by-products), termination by which radicals form stable products and finally termination, where reaction between radicals and antioxidants (vitamin C and E) give rise to non-radical products and unreactive radicals.

Many recent reviews describes how ROS can react with PUFAs in cell membranes, also schematic representations of moleculaire reactions are known. Examples of earlier research done showed that inducement of antioxidants decreases the level of lipid peroxidation. Devasagayam et al. showed that increased concentration of caffeine, glutathione and ascorbic acid ihibited lipid peroxidation by reducing ROS formation. Measurements were performed in rat microsomes where TBARS (known as MDA equivalent) and LOOH (product of lipid peroxidation) were used as markers. Leuter et al. showed that there was a correleation between an increased concentration of ROS and lipid peroxidation. Leuter et al. performed the study in the brain of rats which where aging overtime. Finally, in a more recent study, resveratrol was used to measure the TBARS level at various concentration. Resveratrol is known to act as an antioxidant in vitro. Nosál et al. showed that a higher concentration of resveratrol leads to a lower level of TBARS, which indirectly showes that a lower concentration of ROS leads to less lipid peroxidation

The link between ROS and lipid peroxidation is biological plausible.

Lipid peroxidation is a general description, the link with ROS is well known but literature also describes the possibility that lipid peroxidation can cause oxidative stress. The product HNE of lipid peroxidation can form protein adducts which can lead to cell damage.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea299138> AOPWiki 2018-04-04T15:03:45

2018-04-05T04:06:52

<upstream-id>4cbe7b00-a84e-412e-9a54-64403998fef7</upstream-id> <downstream-id>eb666a9a-33b3-4b50-a5dd-cebb2a063299</downstream-id> Two main products of lipid peroxidation are MDA and HNE which are highly reactive electrophilic aldehydes. Protein adduct formation by HNE-modification of proteins is the main reaction which occurs in cells after lipid peroxidation. HNE-adducts are also used as markers for lipid peroxidation. There are two main principles of HNE-modification of proteins, the Schiff’s Base Formation and the Michael Addition. Schiff’s Base Formation is the reaction of the aldehydic group of HNE with an amino group of a protein. Where the Michael Addition is a reaction of the HNE double bond to a protein side chain. HNE has the preference for amino acid modification Cys à His à Lys which results in a covalent adduct with the protein nucleophilic side chain.

In the 80s it was already found that HNE can react with proteins and form adducts. Since HNE is a highly reactive electrophilic aldehyde it can easily react with proteins in a timeframe of seconds to minutes. Because of the high reactivity only 1-8% of the HNE formed will interact with proteins, but the number of proteins which are altered lies in the hundreds. Several detection techniques are known to find HNE-adducts, but since some are at low abundance it is hard to find them all. One example is proteomic analysis performed by Andringa et al. After ethanol exposure in rats HNE modified proteins were detected in mitochondria. In a more recent study a direct link was made between lipid peroxidation and protein modifications. With the use of rapid SERS monitoring detection of lipid peroxidation as well as protein modification was performed.

It is known that protein adducts are formed by HNE after lipid peroxidation, so it is biological plausible.

Other aldehyde products of lipid peroxidation can also form protein adducts with proteins. Since HNE is specific for lipid peroxidation it is widely used as marker.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea3254f8> AOPWiki 2018-04-04T15:05:20

2018-04-05T04:18:41

<upstream-id>eb666a9a-33b3-4b50-a5dd-cebb2a063299</upstream-id> <downstream-id>e3e0a61c-43c3-4458-855e-74ea5ba12d17</downstream-id> Covalent binding of metabolites or other molecules, such as HNE, with key ER proteins can induce ER stress or can cause oxidative damage in the ER. The mechanism is not completely understood. The principle is that modified proteins are not able to be folded in the correct way, leading to accumulation of unfolded proteins in the ER. Another possibility is that key proteins in the ER are altered, which inhibits their function. Ultimately the ER homeostasis will be disturbed, which leads to ER stress and the activation of UPR.

HNE-modified proteins are detected after lipid peroxidation, as is described in KER 4. More recent research showed that the modified proteins by HNE are also ER proteins, such as protein disulphide isomerase, glucose regulated protein 58/78 and heat shock protein 60. To confirm whether protein-HNE adducts can induce ER stress and UPR, changes in the PERK pathway were monitored. After HNE treatment in rat aortic smooth muscle cells the expression in the PERK pathway increased. Cumaoglu et al. performed a study related to diabetes, and also found that a higher concentration of HNE in cells lead to a higher expression of PERK. Moreover, other proteasomes function in the cell can be carbonylated by CYP2E1 dependent oxidant stress. For toxicants which can directly form protein adducts which leads to UPR is not much known, certainly not about the mechanism. Cisplatin can bind to microsomal compartments which induce ER stress and ultimately UPR. Metabolites of cyclosporin and acetaminophen can also bind to microsomal compartments with the same effect as cisplatin. Acetaldehyde and malondialdehyde (product lipid peroxidation) can react together and can form MAA-adducts. There is no direct link with UPR, but the MAA-adducts are very stable. MAA modified proteins are found in the lungs and skin. Further research is needed to detect whether the can interact with ER proteins.

The biological plausibility can be found in literature, but mechanism is not known. For ethanol, the metabolite acetaldehyde specifically, there is no direct link between protein adduct and UPR

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea33dad0> AOPWiki 2018-04-04T15:29:58

2018-04-05T04:25:25

<upstream-id>132dff3c-afb4-4c71-a841-a9e99c720064</upstream-id> <downstream-id>e3e0a61c-43c3-4458-855e-74ea5ba12d17</downstream-id> With the increasing ROS concentration and the accumulation of unfolded proteins an UPR is triggered in the ER. This is a defence mechanism in a cell, which starts with ER stress when there is an overload of ROS. At low level the activation of PERK in the UPR can prevent further oxidative stress. In several neurodegenerative diseases ER stress was reported. The exact mechanism and the link between ROS and UPR is not completely understood.

Several studies are performed to link oxidative stress with the UPR. Hayashi et al. showed that ROS, and specifically superoxide, plays a role in inducing ER stress by the UPR. Overexpression of SOD1, an antioxidant, reduces the induction of ATF-4 and CHOP, which are proteins released during UPR. Measurement were performed during ischemic brain injury. Chen et al. looked at the effect of ethanol on ER stress in neuron cells. Ethanol induces ROS formation by CYP2E1 activation, when antioxidant N-acetyl-L-cysteine (NAC) or GSH scavenged the production of ROS there an elimination of the expression of ER markers. Another possibility of ROS generating UPR is the interference of ROS with the folding of proteins in the ER. ROS can stimulate unfolded proteins to transform into misfolding proteins, or inactivate PDI/ERO1 which are responsible for the oxidation of unfolded proteins. Research on steatosis in the liver cells by Tsedensodnom et al. showed that after only after 2 hours of ethanol exposure a UPR response already can be measured. mRNAs levels of Bip, grp94 and dnajc3 were detected, which are known ER stress chaperones. Another pathway of inducing ER stress by UPR is that ROS can cause mitochondrial dysfunction. Cigarette smoke extract in retinal pigmented epithelial leads to accumulation of H2O2 in the mitochondria, which resulted in higher expression of UPR sensors.

There is a link between oxidative stress and the unfolded protein response, but the mechanism is not well understood.

The UPR and ER stress can produce ROS by itself, so inducing a cell with a toxicant can also directly lead to ER stress. Time measurements are important to find out which event occurs first. Also the overall mechanism of this KER is not exactly known.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea4323a0> AOPWiki 2018-04-04T15:30:44

2018-04-05T04:37:40

<upstream-id>4cbe7b00-a84e-412e-9a54-64403998fef7</upstream-id> <downstream-id>3557079d-bdbf-4631-a7f2-7ea69b47cebb</downstream-id>  Lipid peroxidation can induce apoptosis due to two different toxic effects. First of all lipids are responsible for maintaining the integrity of cellular membranes. Due to peroxidation of the lipids in the cellular membrane they lose their composition, structure and dynamics of lipid membranes. Various functions are lost, there is an increase of membrane rigidity, decrease activity of membrane-bound enzymes and altered permeability. Secondly there is the formation of highly reactive compounds such as lipid peroxides (MDA, HNE). These lipid peroxides can generate more ROS or can crosslink with important proteins in the cell. Several apoptosis pathways are started due to increased levels of ROS and HNE. p53 is induced and phosphorylated by HNE, as well as inducement of death receptor Fas (CD95). Also due to lipid peroxidation an energetic disturbance is reached, since the protein pumps lost their function. This can lead to neuronal cell death.

It is biological plausible that lipid peroxidation can lead to apoptosis of cells.

There are two mechanisms of apoptosis induced by HNE (main product of lipid peroxidation), which are the extrinsic and the intrinsic pathway. Extrinsic pathway is triggered by the binding of tumour necrosis factor (TNF) to their death receptors (DR) on the cell surface. HNE can induce extrinsic apoptosis in two different ways, but is mainly activated by Fas aggregation by to HNE adduct formation on the cysteins of the Fas. The promotion of Fas/CD95 DR expression, which belong to the TNF-α family, is induced by HNE. Li et al. showed that a higher concentration in HLE B-3 cells leads to a higher expression of Fas. Furthermore knockout GSTA4 mouse, GSTA4 is an antioxidant for HNE, showed a higher expression of Fas since HNE concentration increased. These studies were performed in different organ tissues of mice. After activation and expression of Fas a pathway is started towards apoptosis by ASK1, JNK an Jun proteins. Jun stimulates the intrinsic apoptotic pathway and stimulate AP-1, pro apoptotic genes, expression after phosphorylation. Sharma et al. showed that an increased HNE concentration leads to a higher expression of ASK1 and JNK. When Fas was inhibited apoptosis was stopped. On the other hand HNE can stimulate a negative feedback loop against apoptosis by stimulating the expression of Daxx, which has a negative effect on ASK1-JNK. In the intrinsic pathway HNE can affect mitochondrial injury, leading to an increased level of Ca2+. This induces apoptotic signals as well as cytochrome c which is released from the mitochondria (known as mitochondrial outer membrane permeabilization (MOMP). Also the redox status of mitochondria can be affected by HNE, leading to mitochondrial crisis and activation of caspases. Liu et al. showed that HNE treatment in Jurkat cells induced caspase 8,3 and 9 activity. Caspase 9 is part of the intrinsic apoptotic pathway. HNE can also cause DNA damage since HNE is genotoxic, which induces activation of p53 in combination with oxidative stress. p53 also interacts with the intrinsic apoptotic pathway. In two more recent studies a more direct link is shown between lipid peroxidation and cell death. With the use of Deuterated Polyunsaturated Fatty Acid (D-PUFA) treatment, which are deuterium-reinforced polyunsaturated fatty acids and are more sensitive against ROS, a significantly decrease in cell death was shown with toxicant inducement. Also a decrease was shown in lipid peroxidation products. D-PUFA works as an inhibitor against lipid peroxidation.

Apoptosis is a mechanism of cell death which occurs during lipid peroxidation. Since cell death is a general term used other mechanisms could also play a role. When enormous levels of ROS and HNE are generated even necrosis can occur. Another form is apoptosis is ferroptosis, which is also linked with lipid peroxidation. Also the defence mechanisms of cells against HNE are not described, which should be taken into account.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea446d00> AOPWiki 2018-04-04T15:37:38

2018-04-05T04:48:15

<upstream-id>e3e0a61c-43c3-4458-855e-74ea5ba12d17</upstream-id> <downstream-id>3557079d-bdbf-4631-a7f2-7ea69b47cebb</downstream-id> During the UPR several proteins are released from the ER. When cells have too much stress, due to ROS or other factors, and can’t restore the ER homeostasis pro-death programs are activated. This is done by the proteins IRE1, PERK and ATF-6 which are released from the ER. The main protein involved in apoptosis is CHOP, which is part of the pathway after activation of the PERK protein. But also IRE1 plays a big role in the cell death regulation.

Several studies are performed using western blotting and inhibition assays to find out which proteins are activated in UPR and which have a role in cell death. CHOP has two different roles, which can induce cell death or has a protective function for survival. CHOP is activated by ATF4, which is first activated by a phosphorylated eIF2a. eIF2a is phosphorylated by an activated PERK. IRE1 and PERK activation leads to higher expression of caspases which induce cell death, by Nf-kB and ATF4 activation respectively. Another study showed a direct link between UPR and apoptosis. DHCR24 inhibited apoptosis by interfering with ER stress which resulted in lower levels of CHOP.

That UPR can induce apoptosis is known, but the exact mechanism is not completely clear since many proteins play a role.

Different forms of ER stress can lead to different UPR reactions.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea4a5698> AOPWiki 2018-04-04T15:37:59

2018-04-05T04:51:11

<upstream-id>3557079d-bdbf-4631-a7f2-7ea69b47cebb</upstream-id> <downstream-id>fb762852-4035-4f9f-a7a5-72fd6108c31b</downstream-id> Neurodegeneration is the loss of neuron cells in the brain. Apoptosis is a form of cell death that can cause neurodegeneration. Neurodegeneration is linked with diseases such as Alzheimer.

Apoptosis is a form of cell death, where neurodegeneration is the loss of neuron cells in the brain.

More AOPs should be developed to link other forms of cell death with neurodegeneration.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42ea510cb8> AOPWiki 2018-04-04T15:38:29

2018-04-05T04:53:30

CYP2E1 activation and formation of protein adducts leading to neurodegeneration

Brendan Ferreri-Hanberry

BY-SA

2.0

The AOP has two different MIEs: protein adduct formation (MIEa) and CYP2E1 activation (MIEb). Protein adduct formation is the interaction between a chemical, or reactive metabolite, and a protein at molecular level. During this interaction a covalent bond is formed which occurs due to the reaction between an electrophilic chemical and the nucleophilic part of a protein. When a chemical forms a covalent bond with a protein the protein is damaged and can loses its function. Acetaldehyde, the metabolite of ethanol, is also one of these chemicals known to form protein adducts. This is why protein adduct formation is added in this AOP based on ethanol. CYP2E1 is one of the enzymes responsible for the metabolism of ethanol, and because of this metabolic activity the MIE in added in this AOP. CYP2E1 participates in the metabolism of endogenous, small and hydrophobic compounds using a oxidation reaction. CYP2E1 is mainly expressed in rat liver cells, but can also be found in rat brain cells. Furthermore, in the human brain CYP2E1 expression is mainly found in the amygdala and prefrontal cortex. At higher concentrations of ethanol the expression of CYP2E1 increases, as well as the activity of CYP2E1 since it has a relatively high Km value for ethanol. In this AOP four different KEs are used, which are oxidative stress (KE1), lipid peroxidation (KE2), unfolded protein response (UPR) (KE3) and apoptosis (KE4). Oxidative stress can be defined as the imbalance between ROS and defence mechanisms against these ROS. ROS levels in a cell can rise which leads to damage by the oxidizing free radicals. Lipid peroxidation is a form of direct damage to lipids in the cell membrane or organelle membranes. The cell membrane will eventually break due to the build-up of all the damage. MDA  and 4-hydroxynonenal (HNE) are two products of lipid peroxidation. UPR is a reaction activated by stress in the endoplasmic reticulum (ER). ER stress can be induced by too much protein folding which reaches a higher level than the folding capacity. Also accumulation of unfolded protein in the ER and protein adducts formation with important endoplasmic proteins can induce ER stress, which activates UPR. The final KE is apoptosis, which is programmed cell death in general. The process of apoptosis is well regulated and several signal proteins are known to induce the apoptotic process.

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<table border="1" cellpadding="0" cellspacing="0"> <tbody> <tr> <td style="width:113px"> Key Event </td> <td style="width:491px"> Essentiality </td> </tr> <tr> <td style="width:113px"> MIEa (Protein Adduct Formation) </td> <td style="width:491px"> Moderate support. Activation of MIEa induces increased activation of KE 3, but direct evidence is not available. One theory about the mechanism is that adducts are formed with critical ER proteins. (Haberzettl, P. & Hill, B. G., 2013; Galligan, J. J. et al., 2014; Cumaoglu, A. et al., 2014; Kessova, I. G. & Cederbaum, A. I., 2005; Huličiak, M. et al., 2012; Sadrieh, N. & Thomas, P. E., 1994; Shin, N. Y. et al., 2007; Sapkota, M. & Wyatt, T. A., 2015; Tuma, D. J., 2002) </td> </tr> <tr> <td style="width:113px"> MIEb (CYP2E1 Activation) </td> <td style="width:491px"> High support. Direct evidence is available which prevents the upstream KE 1. CYP2E1 knockout as well as inhibition studies are performed. Activation of CYP2E1 by stressors also showed an increased. (Valencia-Olvera, A. C. et al., 2014; Haorah, J. et al, 2008; Luo, J., 2014; Yang, L. & Cederbaum, A., 2011; Lakshman, M. R. et al., 2013; Jimenez-Lopez, J. M. & Cederbaum, A. I., 2005; Gonzalez, F. J., 2005; Albano, E. et al., 1996; Albano, E., 2006; Wu, D. et al., 2012; Cederbaum, A. I., 2010; Lu, Y. et al., 2010; Oneta, C. M. et al., 2002; Lieber, C. S., 2004; Emerit, J. et al., 2004) </td> </tr> <tr> <td style="width:113px"> KE 1 (Oxidative Stress) </td> <td style="width:491px"> High support. Direct and indirect evidence is available for the essentiality of KE 1. Blocking ROS formation inhibits upstream KE 2 and KE 3. The indirect evidence showed that higher ROS induction showed an increased activity of upstream KE 2 and KE 3. </td> </tr> <tr> <td style="width:113px"> KE 2 (Lipid Peroxidation) </td> <td style="width:491px"> High support. There is much indirect evidence available showing that inducement of lipid peroxidation can increase activity of MIEb and KE 4. The direct evidence of blocking HNE which results in inhibition of upstream KE 4 shows that there is link between KE 2 and KE 4 and that the underlying molecular pathway is known. </td> </tr> <tr> <td style="width:113px"> KE 3 (UPR) </td> <td style="width:491px"> Moderate support. There is direct as well as indirect evidence available which shows molecular understanding of how KE 3 can induce KE 4. The uncertainty lies in whether ER stress alone can induce KE 4, or that KE 1 also plays a role in it. This is more discussed in detail in chapter 4. </td> </tr> <tr> <td style="width:113px"> KE 4 (Apoptosis) </td> <td style="width:491px"> High support. Neurodegeneration is the loss of neuron cells in the brain. </td> </tr> </tbody> </table> See table below where an overview is provided of the direct and indirect evidence. For the meaning of numbering see Abstract and the image of the AOP, <table border="1" cellpadding="0" cellspacing="0"> <tbody> <tr> <td style="width:76px"> Key event relatio-nship </td> <td style="width:274px"> Methods </td> <td style="width:134px"> Influence on downstream Key events </td> <td style="width:121px"> Direct/Indirect evidence </td> </tr> <tr> <td style="width:76px"> KER 1: MIEb ---> KE1   </td> <td style="width:274px"> 1. Stimulation of CYP2E1 by stressors in rat livers. 2. Inhibition studies of CYP2E1 in neuron cells. 3. CYP2E1 KO in mice where TBARS values are measured. 4. Induction of CYP2E1 results in higher ROS levels an higher CYP2E1 expression, study performed in granule neuron cells. </td> <td style="width:134px"> 1. Activation KE 1 2. Inhibition KE 1 3. Inhibition KE 1 4. Activation KE 1 </td> <td style="width:121px"> 1. Indirect 2. Direct 3. Direct 4. Indirect </td> </tr> <tr> <td style="width:76px"> KER 2: KE1 ---> KE2   </td> <td style="width:274px"> 1. Lower ROS level by adding higher concentrations of antioxidants or resveratrol (inhibitor of ROS). TBARS and LOOH product was measured in rat microsomes. 2. Correlation study where higher ROS levels increased lipid peroxidation in aging brains. </td> <td style="width:134px"> 1. Inhibition KE 2 2. Activation KE 2 </td> <td style="width:121px"> 1. Direct 2. Indirect </td> </tr> <tr> <td style="width:76px"> KER 3: KE1 ---> KE3   </td> <td style="width:274px"> 1. Lower ROS levels by overexpression of antioxidant SOD1, NAC or GSH resulted in induction of UPR markers. Measured in neuron cells. 2. Stimulation of ROS formation by ethanol, which induces the UPR response in 2 hours after exposure. </td> <td style="width:134px"> 1. Inhibition KE 3 2. Activation KE 3 </td> <td style="width:121px"> 1. Direct 2. Indirect </td> </tr> <tr> <td style="width:76px"> KER 4: KE2 ---> MIEa   </td> <td style="width:274px"> 1.Proteomic detection techniques for HNE adducts, HNE is a reactive aldehyde product of lipid peroxidation. 2. SERS monitoring detection, showed link between increased lipid peroxidation and increased protein adduct formation. </td> <td style="width:134px"> 1. Activation MIEb 2. Activation MIEb </td> <td style="width:121px"> 1. Indirect 2. Indirect </td> </tr> <tr> <td style="width:76px"> KER 5: MIEa ---> KE3   </td> <td style="width:274px"> 1. HNE (known to form protein adducts) treatment in rat aortic smooth muscle cells induced expression of the PERK pathway, which is part of the UPR. Same study is also performed in different settings. 2. Some toxicants can form protein adducts with ER proteins, what can induce ER stress and the UPR. </td> <td style="width:134px"> 1. Activation KE 3 2. Activation KE 3 </td> <td style="width:121px"> 1. Indirect 2. Indirect </td> </tr> <tr> <td style="width:76px"> KER 6: KE2 ---> KE4   </td> <td style="width:274px"> 1. HNE can induce Fas/CD95DR expression, which regulated the extrinsic pathway of apoptosis. 2. Knockout of GSTA4 in mouse, which is an antioxidant for HNE, showed an increase in Fas expression. 3. ASK1 and JNK are activated by Fas. Increased HNE concentrations showed higher expression of ASK1 and JNK. When Fas was inhibited apoptosis was stopped. 4. HNE induces mitochondrial dysfunction which leads to apoptosis. Higher HNE levels showed increased expression of cytochrome c and caspases. Caspase 3 and 9 are mainly activated. Both are part of the intrinsic pathway of apoptosis. </td> <td style="width:134px"> 1. Activation KE 4 2. Inhibition KE 4 3. Activation KE 4 4. Activation KE 4 </td> <td style="width:121px"> 1. Indirect 2. Direct 3. Indirect 4. Indirect </td> </tr> <tr> <td style="width:76px"> KER 7: KE3 ---> KE4   </td> <td style="width:274px"> 1. Higher expression of IRE1 and PERK, which are UPR markers, showed an increase of caspases expression. These caspases play a major role in the apoptotic pathway. 2. Inhibition of ER stress by DHCR24 resulted in a lower level of CHOP expression. Also an inhibition of apoptosis was shown. </td> <td style="width:134px"> 1. Activation KE 4 2. Inhibition KE 4 </td> <td style="width:121px"> 1. Indirect 2. Direct </td> </tr> <tr> <td style="width:76px"> KER 8: KE4 ---> AO </td> <td style="width:274px"> 1. Neuron loss is detected in neurodegenerative diseases, such as Alzheimer. </td> <td style="width:134px"> 1. Activation AO </td> <td style="width:121px"> 1. Indirect </td> </tr> </tbody> </table>

In AOP1 there are some knowledge gaps present which is one of the principles of the AOP concept. CYP2E1 activation is known to increase the ROS concentration in a cell, but the underlying mechanism is not completely understood. There are two main mechanisms which are suggested in literature, either CYP2E1 or NADPH oxidase could be the primary enzyme which is responsible for ROS formation and cause the further damage in the cells. NAPDH oxidase recycles the NADP+ which is formed during the reaction cycle of CYP2E1, during this cycle ROS is formed due to the uncoupling reaction. CYP2E1 shows a relatively high activity of NADPH oxidase activity and is poorly coupled with NADPH-cytochrome P450 reductase. When NADPH oxidase is inhibited by anti-CYP2E1 IgG a reduction of ROS induced lipid peroxidation was shown. Knock-out or inhibition of CYP2E1 itself resulted in lower oxidative stress. A study performed by Bradford et al. showed that NADPH oxidase knock-out mice attenuated liver injury, where CYP2E1 knock-out mice did not show attenuating of liver injury. On the other hand, NADHP oxidase knock-out mice did not reduce oxidative stress damage to DNA, where CYP2E1 knock-out mice did reduced the damage. Another study by Zhang et al. looked at the influence of NADPH oxidase, an inhibiter against NADPH oxidase was used which reduced the formation of ROS in PC12 cells. Finally, Shah et al. and Furukawa et al. also showed that NADPH oxidase inhibition leads to a reduced formation of ROS, both studies were done in different disease context. The principle of ROS formation by NADPH oxidase is the formation of H2O2 since O2 is used as a substrate. By the Fenton-Weiss-Haber reaction multiple oxidants can be produces. But as mentioned above, several studies showed that CYP2E1 inhibition alone is enough to reduce ROS formation. To take into account, studies described above are all done in liver cells. The mechanism of CYP2E1 activation could be different in the brain. Another knowledge gap is the mechanism of protein adducts that can induce ER stress, and ultimately the UPR. The assumed mechanism is that protein adducts are formed with critical ER proteins, which leads to the dysfunction of the ER. Furthermore, it is also a possibility that protein adducts inhibit the folding of proteins. These proteins can accumulate in the ER and when the protein accumulation is higher than the capacity ER stress is induced. Further research must be done to define the mechanism of how ER stress is induced by protein adducts, which will eventually lead to the UPR.

Not Specified

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