Uticaj tretmana akrilamidom na endokrini pankreas pacova

Abstract

Akrilamid 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.

Acrylamide 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.