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Effect of acrylamide treatment on endocrine pancreas of the rats

dc.contributor.advisorMarković, Jelena
dc.contributor.otherMatavulj, Milica
dc.contributor.otherMarković, Jelena
dc.contributor.otherMilošević, Verica
dc.contributor.otherKojić, Danijela
dc.contributor.otherUšćebrka, Gordana
dc.contributor.otherVojinović-Miloradov, Mirjana
dc.creatorStošić, Milena
dc.date.accessioned2018-06-27T10:19:33Z
dc.date.available2018-06-27T10:19:33Z
dc.date.available2020-07-03T13:47:03Z
dc.date.issued2018-06-22
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/9546
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/Disertacija151989164779197.pdf?controlNumber=(BISIS)107157&fileName=151989164779197.pdf&id=11033&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/record.jsf?recordId=107157&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/IzvestajKomisije151989183918632.pdf?controlNumber=(BISIS)107157&fileName=151989183918632.pdf&id=11034&source=NaRDuS&language=srsr
dc.description.abstractAkrilamid  je toksična hemijska supst anca koja je već dugi niz godina prisutna u životnoj sredini,  jer se kao važan monomer koristi u različite industrijske i laboratorijske svrhe. U poslednjih petnaest godina, akrilamid je postao posebno zanimljiv za šire naučne krugove jer  se pokazalo da  se  nalazi  i u  hrani  biljnog porekla, posebno hrani bogatoj skrobom, koja se priprema pečenjem ili prženjem na temperaturama višim od 120°C.  Do sada ustanovljeni negativni zdravstveni efekti akrilamida su veoma raznovrsni i mogu biti rezultat delovanja samog  akrilamida ili delovanja njegovog metabolita glicidamida koji nastaje  in vivo  kada se jedan deo molekula akrilamida metaboliše oksigenacijom dvostruke veze pomoću enzima citohrom P450 2E1 (CYP2E1). Akrilamid je supstanca koja ima dokazan negativan efekat  na organske sisteme kod ljudi i životinja, i koja je svrstana u moguće humane karcinogene. Negativan efekat akrilamida na egzokrini pankreas je poznat, ali o mogućim efektima akrilamida na endokrini pankreas se i dalje veoma malo zna. Ima puno dokaza koji  ukazuju na to da akrilamid ima citotoksični efekat koji se  manifestuje kroz uticaj na redoks-status ćelija i dovodi do promena u vrednostima biomarkera oksidativnog i nitrozativnog stresa, kao i u aktivnosti antioksidativnih enzima. Pankreas  je  jedan od ciljnih  organa za delovanje akrilamida te je  glavni predmet istraživanja  doktorske teze  bio proučavanje potencijalnog efekta akrilamida na endokrini pankreas pacova.  Ispitivanje je vršeno na 3  eksperimentalne grupe  juvenilnih  mužjaka pacova soja Wistar,  od kojih je  jedna grupa bila kontrolna, dok su dve bile tretirane  sa akrilamidom u dozama od 25 mg/kg tm i 50 mg/kg tm,  5 dana nedeljno,  tokom 3 nedelje. Po isteku tretmana,  nakon dekapitacije, kompletno tkivo pankreasa  je  fiksirano u 10% rastvoru formalina  tokom  24  h i obrađeno prema  standardnoj proceduri za kalupljenje u parafinu.  Parafinski kalupi su sečeni na serijske preseke debljine 5 µm, nakon čega su bojeni  histohemijskom i imunohistohemijskim metodama.  Kod eksperimentalnih grupa posmatrane  su  histološke promene na endokrinom pankreasu, sa akcentom na α-  i β-ćelije.  Takođe, posmatrana je  i  ekspresija  hormona insulina i glukagona, enzima inducibilne azot -oksi d  sintetaze (iNOS) i  CYP2E1,  kao  i ekspresija   antioksidativnih enzima  katalaza  (CAT) i superoksid dismut aza 1 i 2  (SOD1 i SOD2)  u ćelijama Langerhansovih ostrvaca. Potencijalna promena u funkcionalnosti β-ćelija je ispitana i kroz analizu nivoa glukoze u serumu pacova tretiranih sa akrilamidom. Budući da β-ćelije čine 80% ćelija koje grade Langerhansova ostrvca pankreasa,  pored in vivo  eksperimenata, ispitana  je  i toksičnost akrilamida na  Rin-5F ćelijsku liniju insulinoma β-ćelija pacova u in vitro uslovima. Glavni cilj in vitro  istraživanja je bio  da se  ispita  uticaj  rastućih  koncentracija akrilamida na preživljavanje tretiranih  Rin-5F  ćelija, ali i efekat IC50  koncentracije ove supstance primenjene  tokom  različitih vremenskih intervala  (0,5, 1, 3, 6, 12 i 24 h)  na pojavu oksidativnog i nitrozativnog stresa. Redoks-status Rin-5F ćelija tretiranih  sa akrilamidom je ispitan preko analize prisustva biomarkera oksidativnog i nitrozativnog stresa, akrivnosti CAT i ukupne SOD, kao i promene u ekspresiji gena za CAT, SOD1, SOD2   i iNOS.  Pored toga, analiziran je i efekat istog tretmana na  ekspresiju gena za insulin, CYP2E1, Bax i Bcl-2. U okviru teze je pokazano da akrilamid ne dovodi do  značajnih promena u histološkoj građi, dijametru i broju Langerhansovih ostrvaca  kod  tretiranih životinja.  Primena stereoloških metoda  je  ukazala  na mikrostrukturne promene na  endokrinom pankreasu na nivou α-  i β-ćelija. U ovoj tezi je po prvi put pokazano da tretman akrilamidom negativno utiče na broj i površinu β-ćelija pankreasa.  U tezi je, takođe,  pokazan  značajan dozno-zavisni pad u prisustvu insulina u β-ćelijama   pankreasa. Uprkos  tome, kod  akrilamidom tretiranih  životinja  nije konstatovana  promena  u  koncentraciji serumske glukoze.  U  ovoj tezi je pokazano da tretman akrilamidom dovodi do   statistički značajnog porasta  u broju α-ćelija  kod životinja koje su primale nižu dozu tretmana, dok se  broj α-ćelija  kod životinja koje su primale višu dozu tretmana  ne razlikuje značajno od kontrole.  Tretman akrilamidom je doveo do značajnog  porasta u količini   prisutnog glukagona  u α-ćelijama pankreasa. Tretman akrilamidom nije doveo do značajne promene u ekspresiji CAT, SOD1 i SOD2 u ćelijama Langerhansovih ostrvaca.  Kod  tretiranih životinja  došlo do značajnog dozno-zavisnog porasta  u ekspresiji  enzima iNOS,  dok je ekspresija  CYP2E1 značajno dozno-zavisno opala  nakon tretmana. U  tezi je pokazano da tretman akrilamidom negativno utiče na vijabilnost Rin-5F ćelija, i utvrđeno je da IC50  koncentracija akrilamida za Rin-5F ćelije iznosi 10 mM.  Rezultati teze pokazuju da tretman akrilamidom u IC50  koncentraciji u Rin-5F ćelijskoj liniji značajno povećava nivo malondialdehida (MDA) nakon tretmana u trajanju od 1, 12 i 24 h.  Isti tretman  značajno smanjuje nivo redukovanog GSH nakon tretmana od 1, 3, 6, 12 i 24 h, kao i nivo slobodnih  –SH grupa nakon tretmana od 3 i 6 h. Tretman akrilamidom u IC50  koncentraciji signifikantno pojačava aktivnost CAT nakon tretmana od 1 h, dok tretman u trajanju od 12 h značajno smanjuje aktivnost ovog enzima. Ovaj tretman smanjuje aktivnost SOD nakon 1, 12 i 24 h, dok  tretman u trajanju od 6 h značajno pojačava aktivnost enzima SOD.  U tezi je, takođe, pokazan i veoma značajan porast  u nivou prisutnih nitrita,  koji  je direktno proporcionalan  sa nivoom azot-oksida i nivoom akivnosti enzima iNOS.  Ovaj  nalaz ukazuje na potencijalnu pojavu nitrozati vnog stresa u akrilamidom-tretiranim Rin-5F ćelijama.  U  tezi je po prvi put pokazano da tretman  akrilamidom dovodi do  značajnih  varijacija  u transkripciji gena za iNOS, SOD1, SOD2,  CAT,  CYP2E1,  Bax i Bcl-2 u tretiranim Rin-5F ćelijama, dok isti tretman ne dovodi do  promene nivoa  transkripcije gena za insulin.  Tretman akrilamidom u koncentraciji od 10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK gena za iNOS u svim tačkama tretmana, do porasta  nivoa  iRNK za SOD1 i SOD2 nakon tretmana od 12 i 24 h, kao i do porasta  količine  iRNK za CAT nakon tretmana od 3 h.  U  tezi je pokazano  i  da akrilamid  izaziva  promene  u sintezi  iRNK  za enzim  CYP2E1  koji je  posebno značajan u kontekstu detoksikacije ove toksične supstance.  Porast u transkripciji gena za  CYP2E1  je uočen  nakon tretmana u trajanju od 0,5 i 1 h, dok je  do smanjenja transkripcije  došlo  nakon tretmana od 12  i 24  h.  Tretman akrilamidom u koncentraciji od  10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK  gena za Bax u svim tačkama tretmana, i do porasta u transkripciji gena za Bcl-2 nakon tretmana od 0,5, 1 i 3 h. Sumirajući  sve  rezultate  ove teze,  moze se zaključiti  da je endokrini pankreas  jedno od  ciljnih tkiva, na koje akrilamid ostvaruje višestruki negativni uticaj.sr
dc.description.abstractAcrylamide is a toxic chemical used as an important monomer for various industrial and laboratory purposes, which makes it highly present in the environment. In the last fifteen years, acrylamide has become especially interesting for wider scientific circles when it was found in staple foodstuff rich in starch, prepared at temperatures higher than 120°C. The established negative health effects of acrylamide are very diverse and can be the result of the acrylamide action itself or the action of its metabolite glycidamide that occurs in vivo, when acrylamide molecule is metabolized via oxygenation of the double bond by the cytochrome P450 2E1 (CYP2E1). Acrylamide is a substance with a proven adverse effect on humans and animals, and it is classified as a possible human carcinogen. The negative effect of acrylamide on the exocrine pancreas has already been recognized, but the possible effects of acrylamide  on endocrine pancreas are still mostly undetermined. There is a significant amount of evidence to suggest that acrylamide exerts a cytotoxic effect which manifests through the changes in level of oxidative and nitrosative stress biomarkers, as well as in the activity of antioxidant enzymes. Since, pancreas is one of the target organs for acrylamide, the main subject of doctoral thesis was to investigate the potential effect of acrylamide on the rat endocrine pancreas. The investigation was conducted on 3 experimental groups of juvenile male Wistar rats, of which one group was the control group, while two groups were treated with acrylamide at doses of 25 mg/kg bw and 50 mg/kg bw, 5 days a week, during 3 weeks. After termination of the treatment, decapitation was performed, and the complete pancreatic tissue was fixed in a 10% formalin solution for 24 h and treated according to the standard paraffin embedding procedure. Paraffin molds were cut into 5 μm thick serial sections, after which they were stained with histochemical and immunohistochemical methods. Histological changes ofthe endocrine pancreas, with the emphasis on α- and β-cells, were examined in three experimental groups of rats. In addition, the expression of insulin and glucagon hormone, the inducible nitric oxide synthase (iNOS) and CYP2E1 enzymes, and the expression of antioxidative enzymes catalase (CAT) and superoxide dismutases 1 and 2  (SOD1 and SOD2) in the islets of Langerhans were also investigated. A potential change in the functionality of β-cells was also examined by analyzing glucose level in the serum of rats treated with acrylamide. In pancreatic islets of Langerhans the majority of cells (>80%) are β-cells. Therefore, in addition to in vivo experiments, the toxicity of acrylamide was examined in vitro on rat insulinoma Rin-5F cell line.The main goal of in vitro research was to investigate the impact of increasing acrylamide concentrations on the viability of treated Rin-5F cells, and also to examine whether IC50 concentration of this substance, applied at different intervals of time (0.5, 1, 3, 6, 12 and 24 h), induce oxidative and nitrosative stress. Redox-status of Rin-5F cells treated with acrylamide was examined by analyzing oxidative and nitrosative stress biomarkers, CAT and total SOD activity, as well as changes in the expression of the CAT, SOD1, SOD2 and iNOS. In addition, the effect of the same treatment on the transcription of the insulin, CYP2E1, Bax and Bcl-2 gene was analyzed.The results of the thesis showed that acrylamide treatment does not lead to significant changes in the histological structure, diameter and number of islets of Langerhans of treated animals. Application of stereological methods indicated microstructural changes of α- and β-cells ofendocrine pancreas. It has been shown for the first time that treatment with acrylamide negatively affects the number and surface area of pancreatic β-cells. In addition, a significant dose-dependent decline in the amount of insulin in pancreatic β-cells was also demonstrated. However, no change in serum glucose level was observed in treated animals. Acrylamide treatment led to a statistically significant increase in the number of α-cells in animals receiving a lower dose of treatment, while the number of α-cells in animals receiving a higher dose of treatment did not differ significantly from the control. Treatment with acrylamide led to a significant increase in the amount of the glucagon in α-cells. Treatment with acrylamide did not cause a significant change in the expression of CAT, SOD1 and SOD2 in islets of Langerhans. However, there was a significant dosedependent increase in the  expression of iNOS enzyme, whereas expression of CYP2E1 significantly decreased in dose-dependent manner in treated animals. Results of the thesis showed that acrylamide exerts a negative effect on the viability of Rin-5F cell line. It has been established that the IC50 concentration of acrylamide for the Rin-5F cell line is 10 mM. The results of the thesis indicate that treatment of Rin-5F cell line with IC50 concentration of acrylamide for 1, 12, and 24 h significantly increased the level of malondialdehyde (MDA). Exposure to acrylamide for 1, 3, 6, 12 and 24 h significantly decreased the level of reduced GSH, while the level of free -SH groups was reduced after 3 and 6 h of acrylamide treatments. Treatment with IC50 concentration of acrylamide significantly enhanced CAT activity after 1 h of acrylamide exposure, while 12 h exposure significantly reduced the activity of this enzyme. Application of acrylamide reduced SOD activity after 1, 12, and 24 h exposure, while 6 h exposure significantly increased the activity of SOD enzymes. Results of the thesis also showed a very significant increase of the nitrite level, which is directly proportional to the level of nitrogen oxide (NO) and the level of the iNOS activity. This finding points to the potential occurrence of nitrosative stress in acrylamide-treated Rin-5F cells. It has been shown for the first time that acrylamide treatment leads to significant variations in transcription of iNOS, SOD1, SOD2, CAT, CYP2E1, Bax and Bcl-2 genes in treated Rin-5F cells, while the same treatment does not affect transcription of the insulin gene. Treatment with acrylamide at a concentration of 10 mM for increasing periods of time leads to an increase in the relative amount of the iNOS gene iRNA at all treatment points. Twelve and and 24 h of acrylamide exposure increased the transcription of the SOD1 and SOD2 genes. Transcription of CAT gene was increased after 3 h  ofacrylamide exposure. Furthermore, it has been shown that acrylamide treatment leads to variations in the mRNA synthesis of CYP2E1 gene, which is particularly significant in the context of detoxification of this toxic substance. An increase in the transcription ofthe CYP2E1  gene was observed after 0.5 and 1 h of acrylamide exposure, while the reduction of  transcription occurred after 12 and 24 h of acrylamide exposure. The treatment with 10 mM acrylamide has led to an increase of the transcription of the Bax gene at all treatment points, and also to an increase of transcription of the Bcl-2 gene after of 0.5, 1, and 3 h of acrylamide exposure. Summarizing all the results of this thesis, it can be concluded that the endocrine pancreas  is one of the target tissues of acrylamide, to which this substance exerts a multiple adverse effects.en
dc.languagesr (latin script)
dc.publisherУниверзитет у Новом Саду, Природно-математички факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46009/RS//
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172050/RS//
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-sa/4.0/
dc.sourceУниверзитет у Новом Садуsr
dc.subjectakrilamidsr
dc.subjectacrylamideen
dc.subjectLangerhansova ostrvcasr
dc.subjectpankreassr
dc.subjectinsulinsr
dc.subjectglukagonsr
dc.subjectiNOSsr
dc.subjectCYP2E1sr
dc.subjectSOD1sr
dc.subjectSOD2sr
dc.subjectCATsr
dc.subjectoksidativni stressr
dc.subjectislets of Langerhansen
dc.subjectpancreasen
dc.subjectinsulinen
dc.subjectglucagonen
dc.subjectiNOSen
dc.subjectCYP2E1en
dc.subjectSOD1en
dc.subjectSOD2en
dc.subjectCATen
dc.subjectoxidative stressen
dc.titleUticaj tretmana akrilamidom na endokrini pankreas pacovasr
dc.title.alternativeEffect of acrylamide treatment on endocrine pancreas of the ratsen
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-SA
dcterms.abstractМарковић, Јелена; Матавуљ, Милица; Војиновић-Милорадов, Мирјана; Ушћебрка, Гордана; Којић, Данијела; Милошевић, Верица; Марковић, Јелена; Стошић, Милена; Утицај третмана акриламидом на ендокрини панкреас пацова; Утицај третмана акриламидом на ендокрини панкреас пацова;
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/39060/IzvestajKomisije.pdf
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/39059/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/39059/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/39060/IzvestajKomisije.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_9546


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