STABLE COSMETIC PREPARATION
WIPO Patent Application WO/
The invention relates to a cosmetic preparation containing a) polysilicone-25 and b) 2-ethylhexyl-2-cyano-3,3-diphenylacrylate (octocrylene).
Inventors:
MATHIEU, Claire (Glücksburgerstrasse 17, Hamburg, 22769, DE)
K?HLER, Manuela (Hermann-Balk-Strasse 113, Hamburg, 22147, DE)
Application Number:
Publication Date:
12/04/2014
Filing Date:
05/13/2014
Export Citation:
BEIERSDORF AG (Unnastrasse 48, Hamburg, 20253, DE)
International Classes:
A61K8/40; A61K8/898; A61Q17/04
View Patent Images:
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Foreign References:
Other References:
[Online] 7. August -08-07), "W/O emulsifier for suncare", XP, Database accession no. IPCOMD
Anonymous:
"SILFORM* EOF emulsifier EO-free W/O-Emulsifier (Marketing Bulletin)", Momentive , 31. Dezember -12-31), Seiten 1-12, XP, Gefunden im Internet: URL:http://www.google.de/url?url=/workarea/downloadasset.aspx%3Fid%3D26563&rct=j&frm=1&q=&esrc=s&sa=U&ei=DbbQU-itGKKi4gTHnoHwCQ&ved=0CBoQFjAB&sig2=p_OtbwPzjDSlyTGOU9jxPA&usg=AFQjCNE9iQV1m8H23gQFfFtIatK3tRQjkA [gefunden am ]
Patentansprüche
Kosmetische Zubereitung enthaltend
a) Polysilicone-25 und
b) 2-Ethylhexyl-2-cyano-3,3-diphenylacrylat (Octocrylene).
Kosmetische Zubereitung nach Anspruch 1 , dadurch gekennzeichnet, dass die Zubereitung Polysilicone-25 in einer Konzentration von 0,1 bis 2,0 Gewichts-%, bezogen auf das Gesamtgewicht der Zubereitung, enth?lt.
Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Zubereitung 2-Ethylhexyl-2-cyano-3,3-diphenylacrylat in einer Konzentration von 2 bis 12 Gewichts-%, bezogen auf das Gesamtgewicht der Zusammensetzung, enth?lt.
Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Zubereitung einen oder mehrere UV-Filter enth?lt, gew?hlt aus der Gruppe der Verbindungen 2-Phenylbenzimidazol-5-sulfons?ure und/oder deren S Phenylen-1 ,4-bis-(2-benzimidazyl)-3,3'-5,5'-tetrasulfons? 1 ,4- di(2-oxo-10-Sulfo-3-bornylidenmethyl)-Benzol und dessen S 4-(2-Oxo-3-bornyli- denmethyl)benzolsulfons? 2-Methyl-5-(2-oxo-3-bornylidenmethyl)sulfon- s? 2,2'-Methylen-bis-(6-(2H-benzotriazol-2-yl)-4-(1 ,1 ,3,3-tetramethylbutyl)- phenol); 2-(2H-benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-[1 ,3,3,3-tetramethyl-1 -[(tri- methylsilyl)oxy]disiloxanyl]propyl]- 3-(4-Methylbenzyliden) 3-Benzyli- E Terephthalidendicamphersulfons? 4-(tert.- Butyl)-4'-me 4-(Dimethylamino)-benzoes?ure(2-ethylhexyl)- 4-(Dimethylamino)benzoes?ure- 4-Methoxybenzalmalon-s?uredi(2- ethylhexyl) 4-Methoxyzimts?ure(2-ethylhexyl) 4-Methoxyzimts?ureiso- 2-Hydroxy-4-methoxybenzophenon, 2-Hydroxy-4-methoxy-4'- 2,2'-Dihydroxy-4- 2-(4'-Diethylamino-2'- hydoxybenzoyl)-benzoes? H 2-Ethylhexyl-2- Dimethico 3-(4-(2,2-bis Ethoxycarbonylvinyl)- phenoxy)propenyl)-methoxysiloxan / Dimethylsiloxan - C
Dioctylbutylamidotriazon (INCI: Diethylhexyl-Butamidotriazone); 2,4-bis-[5-1 (dimethyl- propyl)benzoxazol-2-yl-(4-phenyl)-imino]-6-(2-ethylhexyl)-imino-1 ,3,5-triazin mit der (CAS Nr. -0); 4,4',4"-(1 ,3,5-Triazin-2,4,6-triyltriimino)-tris-benzoes?ure-tris- (2-ethylhexylester) (auch: 2,4,6-Tris-[anilino-(p-carbo-2'-ethyl-1 '-hexyloxy)]-1 ,3,5-triazin (INCI: Ethylhexyl Triazone); 2,4-Bis-{[4-(2-ethyl-hexyloxy)-2-hydroxy]-phenyl}-6- (4-methoxyphenyl)-1 ,3,5- 2,4,6-Tribiphenyl-4-yl-1 ,3,5- Merocyanine.
5. Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Zubereitung einen oder mehrere Wirkstoffe gew?hlt aus der Gruppe der Verbindungen Gylcyrrhetins?ure, Harnstoff, Arctiin, alpha-Lipons?ure, Fols?ure, Phytoen, D-Biotin, Coenzym Q10, alpha-Glucosylrutin, Carnitin, Carnosin, Coffein, natürliche und/oder synthetische Isoflavonoide, Glycerylglucose, Kreatin, Kreatinin, Taurin, ss-Alanin und/oder Licochalcon A enth?lt.
6. Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch
gekennzeichnet, dass die Zubereitung Propylenglycol, Butylenglycol, 2- Methylpropan-1 ,3-diol, 1 ,2-Pentandiol, 1 ,2-Hexandiol, 1 ,2-Octandiol und/oder 1 ,2- Decandiol enth?lt.
7. Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch
gekennzeichnet, dass die Zubereitung in Form einer W/O-Emulsion vorliegt.
8. Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch
gekennzeichnet, dass die ?lphase der Zubereitung Cetearylisononanoat, C12-15 Alkylbenzoat und/oder Ethylhexylcocoat enth?lt.
9. Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch
gekennzeichnet, dass die Zubereitung Ethanol enth?lt.
10. Kosmetische Zubereitung nach einem der vorhergehenden Ansprüche, dadurch
gekennzeichnet, dass die Zubereitung frei ist von Parabenen.
Description:
Beschreibung
Stabile kosmetische Zubereitung
Die vorliegende Erfindung betrifft eine kosmetische Zubereitung enthaltend Polysilicone-25 und 2-Ethylhexyl-2-cyano-3,3-diphenylacrylat (Octocrylene).
Der Trend weg von der vornehmen Bl?sse hin zur,,gesunden, sportlich braunen Haut" ist seit Jahren ungebrochen. Um diese zu erzielen setzen die Menschen ihre Haut der
Sonnenstrahlung aus, da diese eine Pigmentbildung im Sinne einer Melaninbildung hervorruft. Die ultraviolette Strahlung des Sonnenlichtes hat jedoch auch eine sch?digende Wirkung auf die Haut. Neben der akuten Sch?digung (Sonnenbrand) treten Langzeitsch?den wie ein erh?htes Risiko an Hautkrebs zu erkranken bei überm?ssiger Bestrahlung mit Licht aus dem UVB-Bereich (Wellenl?nge: 280-320 nm) auf. Die überm?ssige Einwirkung der UVB- und UVA-Strahlung (Wellenl?nge: 320-400 nm) führt darüber hinaus zu einer Schw?chung der elastischen und kollagenen Fasern des Bindegewebes. Dies führt zu zahlreichen phototoxischen und photoallergischen Reaktionen und hat eine vorzeitige Hautalterung zur Folge.
Zum Schutz der Haut wurden daher eine Reihe von Lichtschutzfiltersubstanzen entwickelt, die in kosmetischen Zubereitungen eingesetzt werden k?nnen. Diese UVA- und UVB-Filter sind in den meisten Industriel?ndern in Form von Positivlisten wie der Anlage 7 der deutschen Kosmetikverordnung zusammengefasst.
Die Vielzahl an kommerziell erh?ltlichen Sonnenschutzmitteln darf jedoch nicht darüber hinwegt?uschen, dass diese Zubereitungen des Standes der Technik eine Reihe von Nachteilen aufweisen.
Sonnenschutzmittel werden h?ufig in Zusammenhang mit Wassersportaktivit?ten (Baden, Schwimmen, Surfen, Tauchen etc.) verwendet. Dabei tritt das Problem auf, dass das Sonnenschutzmittel bei Kontakt mit Wasser im Laufe der Zeit von der Haut abgewaschen wird. Der Lichtschutzfaktor auf der Haut geht damit zurück. Die Haut muss daher regelm?ssig mit Sonnenschutzmittel nachbehandelt werden, um den UV-Schutz aufrecht zu erhalten. Um die Anhaftung von Sonnenschutzmitteln auf der Haut zu erh?hen, werden nach dem Stand der Technik den Zubereitungen Filmbildner zugesetzt. Diese führen aber regelm?ssig zu einem klebrigen, sensorisch unattraktiven Hautgefühl.
Es war daher die Aufgabe der vorliegenden Erfindung, die Nachteile des Standes der Technik zu beseitigen und ein Sonnenschutzmittel zu entwickeln, dass auch bei intensiven Kontakt mit Wasser einen hohen UV-Schutz gew?hrleistet. Darüber hinaus sollte dieses Sonnenschutzmittel ein attraktives, nicht klebriges Hautgefühl aufweisen.
?berraschend gel?st wird die Aufgabe durch eine kosmetische Zubereitung enthaltend a) Polysilicone-25 und
b) 2-Ethylhexyl-2-cyano-3,3-diphenylacrylat (Octocrylene).
Insbesondere war es für den Fachmann überraschend und nicht vorhersehbar, dass die Zubereitungen besonders wasserfest sind und bei erfindungsgem?ss besonders bevorzugten Ausführungsformen der gemessene Lichtschutzfaktor SPF nach dem Kontakt mit Wasser h?her ist, als nach dem direkten Auftrag der Zubereitung auf die Haut.
Zwar kennt der Stand der Technik das Marketing Bulletin,,Silform EOF emulsifier EO-free W/O-Emulsifier" der Firma Momentive, doch konnte diese Produktinformation nicht den Weg zur vorliegenden Erfindung weisen.
Es ist erfindungsgem?ss vorteilhaft, wenn die erfindungsgem?sse Zubereitung Polysilicone-25 in einer Konzentration von 0,1 bis 2,0 Gewichts-%, bezogen auf das Gesamtgewicht der Zubereitung, enth?lt.
Es ist erfindungsgem?ss bevorzugt, wenn die erfindungsgem?sse Zubereitung Polysilicone-25 in einer Konzentration von 0,5 bis 1 ,0 Gewichts-%, bezogen auf das Gesamtgewicht der Zubereitung, enth?lt.
Das erfindungsgem?sse Polysilicone-25 kann bei der Firma Momentive unter dem
Handelsnamen SILFORM EOF k?uflich erworben werden.
Es ist erfindungsgem?ss vorteilhaft, wenn die erfindungsgem?sse Zubereitung 2-Ethylhexyl-2- cyano-3,3-diphenylacrylat in einer Konzentration von 2,0 bis 12,0 Gewichts-%, bezogen auf das Gesamtgewicht der Zusammensetzung, enth?lt. Es ist erfindungsgem?ss bevorzugt, wenn die erfindungsgem?sse Zubereitung 2-Ethylhexyl-2- cyano-3,3-diphenylacrylat in einer Konzentration von 5,0 bis 10,0 Gewichts-%, bezogen auf das Gesamtgewicht der Zusammensetzung, enth?lt.
Im Rahmen der vorliegenden Offenbarung ist mit Zubereitung immer die erfindungsgem?sse Zubereitung gemeint, wenn nichts anderes erw?hnt ist.
Es ist erfindungsgem?ss vorteilhaft, wenn die erfindungsgem?sse Zubereitung einen oder mehrere zus?tzliche UV-Filter enth?lt.
Erfindungsgem?ss bevorzugt handelt es sich bei der erfindungsgem?ssen Zubereitung um ein kosmetisches Sonnenschutzmittel.
Es ist erfindungsgem?ss vorteilhaft, wenn die erfindungsgem?sse Zubereitung einen oder mehrere weitere UV-Filter gew?hlt aus der Gruppe der Verbindungen Phenylen-1 ,4-bis-(2- benzimidazyl)- 3,3'-5,5'-tetrasulfons? 2-Phenylbenzimidazol-5-sulfons? 1 ,4-di(2- oxo-10-Sulfo-3-bornylidenmethyl)-Benzol und dessen S 4-(2-Oxo-3- bornylidenmethyl)ben25
zolsulfons? 2-Methyl-5-(2-oxo-3-bornylidenmethyl)sulfons? 2,2'-Methylen- bis- (6-(2H-benzotriazol-2-yl)-4-(1 ,1 ,3,3-tetramethylbutyl)-phenol); 2-(2H-benzotriazol-2-yl)-4- methyl- 6-[2-methyl-3-[1 ,3,3,3-tetramethyl-1 -[(trimethylsilyl)oxy]disiloxanyl]propyl]- 3-(4- Methylbenzyliden) 3-B E 2-(4'-Diethylamino-2'- hydoxybenzoyl)-benzoes? Terephthalidendicamphersulfons? 4- 30 (Dimethylamino)-benzoes?ure(2-ethylhexyl) 4-(Dimethylamino)benzoes?ure- 4- Methoxybenzalmalon-s?uredi(2-ethylhexyl) 4-Methoxyzimts?ure(2-ethylhexyl) 4- Methoxyzimts? 2-Hydroxy-4-methoxybenzophenon, 2-Hydroxy-4-methoxy- 4'- 2,2'-Dihydroxy-4- H 2- Ethylhexyl- 2-; Dimethico 3-(4-(2,2-bis Ethoxycarbonylvinyl)- 35 phenoxy)propenyl)-methoxysiloxan / Dimethylsiloxan - C
Dioctylbutylamidotriazon (INCI: Diethylhexyl-Butamidotriazone); 2,4-bis-[5-1 (dimethylpropyl)benzoxazol-2-yl-(4- phenyl)- imino]-6-(2-ethylhexyl)-imino-1 ,3,5-triazin mit der (CAS Nr. -0); 4,4',4"-(1 ,3,5- Triazin-2,4,6-triyltriimino)-tris-benzoes?ure-tris(2-ethylhexylester) (auch: 2,4,6-Tris-[anilino-(p- carbo-2'- ethyl-1 '-hexyloxy)]-1 ,3,5-triazin (INCI: Ethylhexyl Triazone); 2,4-Bis-{[4-(2-ethyl-hexyloxy)-2- hydroxy]- phenyl}-6-(4-methoxyphenyl)-1 ,3,5-triazin (INCI: Bis-Ethylhexyloxyphenol Methoxyphenyl Triazin); 2,4,6-Tris-(biphenyl)-1 ,3,5- 2,4-Bis-(4'- Di-neopentylaminobenzalmalonat)-6- (4"_
butylaminobenzoat)-5 s-triazin, 4-Dicyanomethylen-2,6-dimethyl-1 ,4-dihydropyridin-N- (ethyloxysulfatestersalz), Titandioxid, Zinkoxid, Merocyanine, Piperazinderivate enth?lt. Erfindungsgem?ss besonders bevorzugte Ausführungsformen der vorliegenden Erfindung sind dadurch gekennzeichnet, dass die Zubereitung frei ist von 3-(4-Methylbenzyliden)- campher und 2-Hydroxy-4-methoxybenzophenon (Oxybenzon).
Hinsichtlich der Einsatzkonzentrationen der UV-Filter ist es erfindungsgem?ss vorteilhaft, wenn die Zubereitung UV-Filter in der Gesamtmenge von 10 bis 40 Gewichts-%, bezogen auf das Gesamtgewicht der Zubereitung, enth?lt. Erfindungsgem?ss bevorzugt ist es, wenn die Zubereitung UV-Filter in der Gesamtmenge von 12 bis 30 Gewichts-%, bezogen auf das Gesamtgewicht der Zubereitung, enth?lt.
Erfindungsgem?ss vorteilhafte Ausführungsformen der vorliegenden Erfindung sind auch dadurch gekennzeichnet, dass die Zubereitung einen oder mehrere Wirkstoffe gew?hlt aus der Gruppe der Verbindungen Gylcyrrhetins?ure, Harnstoff, Arctiin, alpha-Lipons?ure, Fols?ure, Phytoen, D-Biotin, Coenzym Q10, alpha-Glucosylrutin, Carnitin, Carnosin, Coffein, natürliche und/oder synthetische Isoflavonoide, Glycerylglucose, Kreatin, Kreatinin, Taurin, ss-Alanin und/oder Licochalcon A enth?lt.
Erfindungsgem?ss vorteilhaft liegt die erfindungsgem?sse Zubereitung in Form einer W/O- Emulsion vor. Dabei ist es erfindungsgem?ss bevorzugt, wenn die Zubereitung au sser Polysilicone-25 keine weiteren Emulgatoren enth?lt.
Die ?lphase der erfindungsgem?ssen Zubereitung wird vorteilhaft gew?hlt aus der Gruppe der polaren ?le, beispielsweise aus der Gruppe der Lecithine und der Fetts?uretriglyceride, namentlich der Triglycerinester ges?ttigter und/oder unges?ttigter, verzweigter und/oder unverzweigter Alkancarbons?uren einer Kettenl?nge von 8 bis 24, insbesondere 12 bis 18 C- Atomen. Die Fetts?uretriglyceride k?nnen beispielsweise vorteilhaft gew?hlt werden aus der Gruppe der synthetischen, halbsynthetischen und natürlichen ?le, wie z. B. Cocoglycerid, Oliven?l, Sonnenblumen?l, Jojoba?l, Soja?l, Erdnu ss?l, Raps?l, Mandel?l, Palm?l, Kokos?l, Rizinus?l, Weizenkeim?l, Traubenkern?l, Distel?l, Nachtkerzen?l, Macadamianu ss?l und dergleichen mehr.
Erfindungsgem?ss vorteilhaft sind ferner z. B. natürliche Wachse tierischen und pflanzlichen Ursprungs, wie beispielsweise Bienenwachs und andere Insektenwachse sowie
Beerenwachs, Sheabutter und/oder Lanolin (Wollwachs).
Ferner kann die ?lphase vorteilhaft gew?hlt werden aus der Gruppe der Dicaprylylether enthalten.
Es ist ferner vorteilhaft, das oder die Olkomponenten aus der Gruppe Isoparaffine,
Isoeikosan, Neopentylglykoldiheptanoat, Propylenglykoldicaprylat/dicaprat, Capry- lic/Capric/Diglycerylsuccinat, Butylenglykol Dicaprylat/Dicaprat, Ci2-i3-Alkyllactat, Di-C1 2-13- Alkyltartrat, Triisostearin, Dipentaerythrityl Hexacaprylat/Hexacaprat, Propylenglykolmo- noisostearat, Tricaprylin, Dimethylisosorbid.
Vorteilhafte Olkomponenten sind ferner z. B. Butyloctylsalicylat (beispielsweise das unter der Handelsbezeichnung Hallbrite BHB bei der Fa. CP Hall erh?ltliche), Tridecylsalicylat (welches unter der Handelsbezeichnung Cosmacol ESI bei der Fa. Sasol erh?ltlich ist), C1 2- C1 5 Alkylsalicylat (unter der Handelsbezeichnung Dermol NS bei der Fa. Alzo erh?ltlich), Hexadecylbenzoat und Butyloctylbenzoat und Gemische davon (Hallstar AB).
Auch beliebige Abmischungen solcher ?l- und Wachskomponenten sind vorteilhaft im Sinne der vorliegenden Erfindung einzusetzen.
Erfindungsgem?ss bevorzugte Ausführungsformen der vorliegenden Erfindung sind dadurch gekennzeichnet, dass die ?lphase der Zubereitung Cetearylisononanoat, C1 2-1 5
Alkylbenzoat und/oder Ethylhexylcocoat enth?lt.
Erfindungsgem?ss besonders bevorzugt enthaltenden die erfindungsgem?ssen Zubereitungen kein Dimethicone + Dimethicone Crosspolymer.
Die Wasserphase der erfindungsgem?ssen Zubereitungen kann -neben Wasser- vorteilhaft übliche kosmetische Hilfsstoffe enthalten, wie beispielsweise Alkohole, insbesondere solche niedriger C-Zahl wie Ethanol, Isopropanol, Diole oder Polyole niedriger C-Zahl sowie deren Ether, vorzugsweise Propylenglykol, Butylenglykol, 2-Methylpropan-1 ,3-diol, Pentan-1 ,2-diol, Hexan-1 ,2-diol, Octan-1 ,2-diol, Decan-1 ,2-diol, Glycerin, Ethylenglykol, Ethylenglykolmono- ethyl- oder -monobutylether, Propylenglykolmonomethyl, -monoethyl- oder -monobutylether, Diethylenglykolmonomethyl- oder -monoethylether und analoge Produkte, Elektrolyte, etc.. Die erfindungsgem?sse Zubereitung kann vorteilhaft Konsistenzbildner (Gelbildner,
Verdickungsmittel) wie beispielsweise Polyacrylate (auch quervernetzt) oder
Cellulosederivate (beispielsweise Hydroxyethylcellulose) oder andere enthalten.
Es ist erfindungsgem?ss bevorzugt, wenn die erfindungsgem?sse Zubereitung Ethanol enth?lt.
Erfindungsgem?ss bevorzugt ist es, wenn die erfindungsgem?sse Zubereitung dadurch gekennzeichnet ist, dass die Zubereitung Propylenglycol, Butylenglycol, 2-Methylpropan-1 ,3- diol, 1 ,2-Pentandiol, 1 ,2-Hexandiol, 1 ,2-Octandiol und/oder 1 ,2-Decandiol enth?lt. Dabei werden diese Diole/Glycole erfindungsgem?ss vorteilhaft in einer Gesamtmenge von 0,25 bis 4 Gewichts-%, bezogen auf das Gesamtgewicht der Zubereitung, eingesetzt.
Selbstverst?ndlich kann die Zubereitung mit den üblichen, in der Kosmetik verwendeten Konservierungsmitteln konserviert werden.
Dabei ist es erfindungsgem?ss bevorzugt, wenn die Zubereitung Phenoxyethanol enth?lt.
Darüber hinaus ist es erfindungsgem?ss bevorzugt, wenn die Zubereitung frei ist von
Parabenen.
Die nachfolgenden Beispiele sollen die vorliegende Erfindung verdeutlichen, ohne sie einzuschr?nken. Alle Mengenangaben, Anteile und Prozentanteile sind, soweit nicht anders angegeben, auf das Gewicht und die Gesamtmenge bzw. auf das Gesamtgewicht der Zubereitungen bezogen.
& 2004-. All rights reserved.PMCID: PMC4523844Genome-wide hydroxymethylcytosine pattern changes in response to oxidative stress,a,1 ,1 ,1 ,2 ,1 ,1 ,1 ,1 ,3 ,4 ,5 ,4 ,3 ,2 ,5 and
b,12CRUK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, United Kingdom3ULB, Center for Diabetes Research, Faculty of Medicine, ULB, 1070 Brussels, Belgium.4Laboratory of Vaccinology and Mucosal Immunity, Faculty of Medicine, ULB, 1070 Brussels, Belgium.5Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, GermanyaEmail: bEmail:
The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack
models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes
GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of
oxidative stress on the global hydroxymethylome.BSO-treated SY5Y cells and GPx1/2 double-knockout mice have a decreased level of hydroxymethylcytosineTo see whether hmC levels are affected in cells exposed to exogenous oxidative stress, we treated SY5Y cells with BSO and quantified hmC by dot-blot experiments and mass spectrometry (LC/MS/MS) ( and ). SY5Y is a human cell line commonly used as a model for studying oxidative stress and oxidative-stress-related diseases such as Alzheimer’s and Parkinson’s,. Additionally, neuroblastomas are derived from neural crest cells, and neuronal cell types have high levels of hmC, making those cells a good model for our study. BSO inhibits the synthesis of the antioxidant glutathione (GSH) and causes oxidative stress by generating H2O2 ( and ). As shown in , BSO-treated cells were found to have a significant decrease of the global hmC level. Further, this observation appeared not to be attributable to an increased apoptosis or necrosis, as BSO treatment did not increase the proportion of apoptotic or necrotic cells as compared to mock-treated cells ().Oxidative stress reduces the global hydroxymethylcytosine level.Several studies have shown that BSO-treated SY5Y cells display a significant decrease in proliferation in response to oxidative stress,. We therefore measured proliferation with the xCELLigence technology, which records changes in conductivity that are proportional to the number of cells attached to the incubation chamber. Upon treatment with BSO, the proliferation decrease was greater in TET1-knockdown cells than in TET2-knockdown cells or in control cells (). It is worth noting that TET3 does not seem to be expressed in SY5Y cells, and that the effects observed here cannot be attributed to an off-target effect of TET1-RNAi on TET2 (, and data not shown).To confirm and extend the above findings, we used an in vivo model of oxidative stress: double-knockout mice lacking the genes encoding glutathione peroxidases 1 and 2 (called hereafter GPx1/2 Dko). The GPx enzymes are known to reduce H2O2 and are viewed as the major cellular antioxidant enzymes, especially in intestinal epithelial cells where reactive oxygen species accumulate upon their depletion (). Dot-blot and mass spectrometry experiments were performed on GPx1/2 Dko and wild-type (wt) colon epithelia, and in agreement with our data on SY5Y cells, the global hmC level was found to be lower in GPx1/2 Dko mice than in their wt counterparts ().Our results thus suggest that hydroxymethylcytosine levels are decreased upon exogenous and in vivo oxidative assaults, and that SY5Y cells with reduced TET1 expression are more sensitive to oxidative stress.hmC deep-sequencing profiles of BSO-treated SY5Y cells highlight pathways involved in the oxidative stress responseThe global decrease in hmC seen upon treatment of SY5Y cells with BSO () led us to interrogate its genome-wide distribution. For this we used the previously described hmC-selective chemical labeling technique hMe-seal to selectively isolate hydroxymethylated DNA fragments, and subjected these to Illumina deep sequencing (referred here as “hmC-seq”). As previously described (reviewed in), gene bodies appeared most highly represented among the captured fragments, exons being more enriched than introns (). In agreement with the above dot-blot and mass spectrometry data, BSO-treated SY5Y cells displayed a significant global decrease in hmC (, left panel).Genome-wide hmC sequencing after cell treatment with BSO highlights oxidative-stress-related pathways.Despite the observed global decrease of hmC, we next looked at differentially hydroxymethylated genes (termed “dhMGs”) to see if certain genes might locally loose or even gain hmC upon oxidative stress. We found 2846 dhMGs (), 53% of which displayed a local decrease in hmC and 47% a local increase (, right panel). Remarkably, Ingenuity gene ontology analysis applied to these dhMGs revealed significant over-representation of toxicogenomic pathways associated with oxidative stress response, such as mitochondrial dysfunction, decreased polarization of mitochondria, and cytochrome P450 response (). Of note, the most highly over-represented pathways were different according to whether a differentially hydroxymethylated gene showed a gain or a loss of hmC: the mitochondrial dysfunction pathway in the former case and pathways related to the physiopathology of the heart, liver and kidney in the latter ().Interestingly, genes such as BCL2 or the BCL2-related MCL1 gene, found to be differentially hydroxymethylated (; see also sequencing tracks on ), are known to exert important functions during the oxidative stress response: Oxidative stress is attenuated in mice overexpressing BCL2, and the hepatitis B HBx protein sensitizes liver cells to H2O2-induced oxidative stress by reducing MCL1 expression,.Our results thus show that BSO treatment affects the hmC patterns both globally and locally, notably in genes important for a protective response to oxidative stress.Mice lacking the glutathione peroxidases 1 and 2 display an altered hydroxymethylation on genes involved in the oxidative stress responseAs for the above, hmC-seq experiments were performed on GPx1/2 wt and Dko colon epithelia, and confirmed the results obtained with SY5Y cells: mice lacking the GPx genes showed a significant overall decrease in hmC (, left panel); reads most often corresponding to a gene body location and more frequently to an exon than to an intron location ().Mice lacking GPx1/2 display genome-wide hmC changes in genes involved in oxidative-stress-related pathways.We next identified 475 dhMGs (), 81% of which showed a local decrease in hmC and 19%, a local increase (, right panel). Interestingly, a significant proportion of dhMGs were found to be involved in toxicogenomic pathways associated exclusively with oxidative stress, and notably with GSH depletion, precisely emphasizing GPx deletion that occurs in the Dko mice (. In , results are shown separately for genes showing a gain or loss of hmC).As for BSO-treated SY5Y cells, we again found key genes involved in the oxidative stress response. Examples include Nfe2l1/Nrf1 and Prdx6 (), whose products are known, respectively, to initiate transcription of antioxidant genes and to participate in the redox regulation of the cell by reducing H2O2,. It is also worth noting that our pathway analyses did not highlight the same pathways in BSO-treated SY5Y cells and GPx1/2 Dko mice (B and B). This probably reflects the fact that the cultured cells were treated for only 48h, whereas mice lacking the GPx genes were under constant oxidative assault from embryogenesis to the day of sacrifice (1 month).In summary, these results confirm restructuration of the hydroxymethylome, here in response to an in vivo oxidative stress, and uncover a set of genes/pathways that reflect the need for an appropriate cellular response.MicroRNAs encoded by differentially hydroxymethylated genes are predicted to target transcripts involved in the oxidative stress responseDuring our analysis of dhMGs in SY5Y cells and GPx1/2 wt and Dko mice, we observed an unexpected high proportion of differentially hydroxymethylated microRNA-encoding sequences (). MicroRNAs (miRs) are non-coding RNAs of 18 to 24 nucleotides long that hybridize to target mRNAs and, depending on the level of complementarity, cause their degradation or translational repression. As depicted in , 25% of the dhMGs identified in SY5Y cells and 21% of those identified in GPx1/2 Dko mice were found to correspond to miR-encoding sequences (see also ).Differentially hydroxymethylated microRNA genes highlight pathways involved in the oxidative stress response.As many miR targets are conserved among mammalian species, we examined more closely the miR-encoding sequences showing altered hydroxymethylation in BSO-treated SY5Y cells or in GPx1/2 Dko mice. Of these miRs, 20 showed a robust increase or decrease in hmC in both SY5Y cells and in GPx1/2 Dko mice ( and ). Focusing on this set of 20 miRs, we used the miRWalk database, a bioinformatic tool that exploits several prediction programs, to build a comprehensive list of predicted mRNAs targets (, and ). Surprisingly, Ingenuity pathway analysis applied to the corresponding human and mouse mRNAs revealed over-representation of toxicogenomic pathways involved in oxidative stress, as observed earlier (; see also B and B, and ).These results thus suggest that hydroxymethylation pattern changes in DNA regions that do not solely code for proteins (here, microRNA genes), but that may contribute to determine the cellular response to oxidative stress.In this report, we have used different approaches to investigate the links between oxidative stress, the global hmC level, and the local distribution of this epigenetic mark. In vitro and in vivo data on SY5Y cells and in our mouse model show that hmC is significantly decreased upon oxidative stress. In addition, we show that TET1 depletion sensitizes SY5Y cells to oxidative stress induced by BSO, the most commonly used drug to increase intracellular levels of reactive oxygen species (ROS). This suggests that TET1, at least in part, may play a role in protecting cells against oxidative stress.Of note, our genome-wide hmC profiling data shows a global decrease of hmC in both SY5Y cells and in GPx1/2 Dko mice, but locally, some of the differentially hydroxymethylated regions display an increase of hmC in response to oxidative stress. This increase at specific genomic loci might be caused by recruitment of TET enzymes to these regions in order to initiate an appropriate transcriptional program to respond to oxidant assaults.Surprisingly, around 25% of the dhMGs identified here in either SY5Y or mice lie in microRNA-encoding sequences. In silico analyses have revealed that these miRs could target important genes involved in ROS detoxification. MiRs have been shown to target the transcripts of numerous genes that are associated to life-threatening diseases such as neurodegenerative disorders or cancer, and this set of miRs might be exploited, in the future, in biomarker discovery for oxidative stress related diseases,. The present work could thus be the starting point in the development of exciting biomedical applications, once we have gained better understanding of the links between oxidative stress and hydroxymethylcytosine patterns, and between miR-genes hydroxymethylation and the miR-genes transcript levels.Of note, our study is the first that evaluated the impact of oxidative stress in vivo. On the one hand, prolonged oxidative stress is associated with inflammation, which is thought to precede cancer development. In the other hand, a decrease of hmC is now widely accepted as a hallmark of many cancers, linking the TET proteins to cancerogenesis. It is thus tempting to speculate that the decrease of hmC observed during cancer development could be explained, at least partly, by a decrease of hmC emerging during oxidative stress and inflammation.In conclusion, we propose, on the basis of our results, a model linking oxidative stress to hydroxymethylcytosine patterns. Our results notably highlight the unexpected potential role of certain microRNAs in determining how cells respond to oxidative stress. In addition, we expect that these results, in association with other studies, will lift the veil on molecular aspects that could explain the global decrease of hmC in cancers. However, additional investigation on how the TETs are recruited on genes that gain hmC, as well as the transcriptional effects of differential hydroxymethylation on oxidative stress-related genes and microRNAs, still need to be addressed and should be the subject of future studies.Statistical analysisUnless otherwise indicated, all experiments included technical replicates, and were repeated at least three independent times. Data and graphs are presented as averages±standard deviations. Data were compared by means of two-tailed unpaired t-tests for comparison, and statistical significance was accepted at p-values &#x. * and ** represent p-values &#x and &#x, respectively. Statistics relative to hmC deep-sequencing analysis are further described in . Ingenuity p-values were calculated online with Fisher’s exact t-test.Cell culture and treatmentsSY5Y cells (ATCC #CRL- 2266) were maintained in 1:1 Dubelcco’s modified Eagle’s medium (DMEM) and F12 medium, then supplemented with 10% fetal bovine serum and grown at 37°C under 5% CO2. The cells were then treated with 500&#x003M BSO for 48h. The mock-treatment control contained water instead of BSO.GPx1/2-knockout miceIn our in vivo experiments, purified genomic DNA extracts obtained from the colon epithelia of 129 mice with combined disruption of the GPx1 and GPx2 genes were generously provided by Dr. F. F. Chu, and are fully described in. The methods used on mice were carried out in accordance with the approved NIH guidelines, which authorize to perform all of the animal studies described in the related manuscript. In addition, all experimental protocols were approved by the COH Research Animal Care Committee (Duarte, California).Five wild-type and five double-knockout (Dko) mice were used for hmC quantification in dot-blot and mass spectrometry experiments. Of these, two wild-type and two Dko mice were used for subsequent deep-sequencing analysis.Dot blot for 5-hydroxymethylcytosine quantificationDot-blot experiments were done as previously described, with some modifications. For complete procedure, please refer to .Western blot analysesStandard procedures were used for Western blotting. Primary antibodies used in these experiments were directed against TET2 (ab94580; Abcam) or HDAC1 (D pAb-053-050).Analysis of global hmC levels by mass spectrometry (LC/MS/MS)500ng of genomic DNA was incubated with 5 U of DNA Degradase Plus (Zymo Research) at 37°C for 3h. The resulting mixture of 2’-deoxynucleosides was analysed on a Triple Quad 6500 mass spectrometer (AB Sciex) fitted with an Infinity 1290 LC system (Agilent) and an Acquity UPLC HSS T3 column (Waters), using a gradient of water and acetonitrile with 0.1% formic acid. External calibration was performed using synthetic standards, and for accurate quantification, all samples and standards were spiked with isotopically labeled nucleosides. HmC levels are expressed as a percentage of total cytosines.Hydroxymethylated DNA fragment affinity purification (hMe-seal)At least 500ng of genomic DNA was diluted in ultra-pure water to 35ng/&#x003L and then sonicated in cold water with a Bioruptor sonicator (Diagenode) to obtain fragments averaging 300bp in size. The fragmented DNA was used in combination with the hydroxymethyl collector (Active Motif) following to the manufacturer’s protocol. Briefly, a glucose moiety that contains a reactive azide group was enzymatically linked to hydroxymethylcytosine in DNA, creating glucosyl-hydroxymethylcytosine. Next, a biotin conjugate was chemically attached to the modified glucose via a “click reaction”, and magnetic streptavidin beads were used to capture the biotinylated-hmC DNA fragments. After extensive washing steps and chemical elution, the hydroxymethylated DNA fragments released from the beads were used in sequencing experiments.Ingenuity softwareIngenuity IPA software was used for toxicogenomic analysis (“IPA-TOX”). The genes and hmC fold changes were loaded into the Ingenuity database and then core analyses were done, using default parameters and specifying the relevant species (“Human” for SY5Y cells and “Mouse” for mice samples). The tissues used in the experiments were also indicated.For further details on experimental procedures, including, RNA interference and RT-qPCR analysis, dot blot for 5-hydroxymethylcytosine quantification, Reactive Oxygen Species measurements, measures of apoptosis and necrosis, proliferation experiments, library preparation, deep sequencing workflow, and statistical analysis, please refer to .How to cite this article: Delatte, B. et al. Genome-wide hydroxymethylcytosine pattern changes in response to oxidative stress. Sci. Rep.
5, 12714; doi: 10.1038/srep1).Supplementary Information:(3.2M, pdf)BD, JJ, CC, RD are supported by the Belgian FNRS. MD is supported by Innoviris. MB is supported by the Televie. FF is an FNRS ‘Senior Research Associate’. BD is an ‘FNRS Aspirant’. FF’s laboratory was funded by grants from the FNRS and Televie, the ‘Interuniversity Attraction Poles’ (IAP Phase VII no P7/03) and by the ‘Action de Recherche Concerté’ (AUWB- ULB-No 7). We thank Dr. F.F Chu for providing the GPx1/2 Dko and wt genomic DNA extracts.Author Contributions B.D. and F.F. designed experiments and directed the study. B.D., E.C., J.J., C.C., M.R. and T.P.J. performed research and interpreted data. Bioinformatic analyses were conducted by M.D. and M.B.i. L.M. and D.L.E. performed the apoptosis and necrosis measurements. M.L. and F.M. performed the FACS experiments. M.B.a. and A.M. did mass spectrometry experiments. R.D. read and gave input on the manuscript. B.D. and F.F. wrote the manuscript. All authors read and approved the final version of the manuscript.Tahiliani M.
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