Kinik asidin sitotoksik/genotoksik ve antigenotoksik etkilerinin sağlıklı akciğer epitel hücre hatlarında belirlenmesi
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Date
2014-04
Authors
Kocaoğlu, Ersin
Journal Title
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Publisher
Uludağ Üniversitesi
Abstract
Yapılan bu tez çalışmasında kinik asidin sitotoksik ve genotoksik etkileri, insan akciğer epitel hücre hattı (Beas-2b) hücrelerinde klonojenik test, formazan boyama XTT testi ve tek hücre jel elektroforez (Komet) testi kullanılarak araştırılmıştır. Kinik asitin antigenotoksik etkisi ise bilinen bir DNA hasarı ve apoptoz indükleyici kemotarapötik ilaç olan cisplatin ile birlikte kullanılarak çalışılmıştır. Çalışmamızda kinik asidin ve cisplatinin tek başlarına oluşturdukları sitotoksik etkilerin belirlenmesi amacıyla kinik asit için klonojenik test, cisplatin için XTT testi uygulanmıştır. Çeşitli dozlarda kinik asit (12 – 18 μM) ve cisplatin'e (0,5 – 50 μM) maruz bırakılan Beas-2b hücrelerinde hayatta kalış eğrileri hazırlanmıştır. Klonojenik test ile kinik asidin IC50 dozu 15,48 µM olarak belirlenmiştir. XTT testi ile cisplatinin IC50 dozu ise 19 µM olarak belirlenmiştir. Komet testi için kullanılacak optimum kinik asit konsantrasyonları (5 ve 10 μM) ve cisplatin dozları ise IC50 dozu olan 19 µM ve bu dozun yarısı 9,5 µM olarak belirlenmiştir. Komet testi sonucunda kinik asitin 10 µM dozunda genotoksisiteye neden olduğu tespit edilmiştir. Cisplatin ile muamele sonucunda ise 9,5 µM dozunda genotoksisitede anlamlı şekilde artış olduğu gözlenirken, 19 µM dozunda genotoksik etkinin artış göstermediği belirlenmiştir. Kinik asitin antigenotoksik etkisini belirlemek için cisplatinle gerçekleştirilen kombine uygulamalarında ise, hem 9,5 µM hem 19 µM cisplatin iki doz kinik asitle birlikte uygulanmış ve kinik asitin cisplatin uygulamasında gözlenen genotoksik etkiyi azaltmadığı belirlenmiştir. Düşük dozda kinik asit ilave edilen cisplatin uygulamasında komet testinde kuyruk uzunluğu, kuyruk % DNA ve olive kuyruk momenti parametrelerinde anlamlı düşüşler gözlenmiştir. Tek cisplatin dozuna göre oluşan bu düşüşlerin cisplatinin hasarlı hücreleri büyük oranda apoptoza götürmesinden kaynaklandığı düşünülmektedir. Yüksek dozda kinik asitle kombinlenen cisplatin gruplarında ise üç komet parametresinde de anlamlı yükselme gözlenmiştir. Bu durum ise yüksek doz kinik asitin cisplatince oluşturulan apoptozu azaltması ve hasarlı hücre sayısını tekrar yükseltmesinden kaynaklanabilir. Sonuç olarak kinik asit antigenotoksik etki göstermemiştir. Kinik asitin apoptozu baskılama etkisinin olup olmadığı ya da genotoksik etkiye sahip olup olmadığının farklı deney yöntemleri ile araştırılması gerektiğini düşünmekteyiz.
This study investigated the cytotoxic and genotoxic effects of quinic acid alone or its anti-genotoxic effects in combination with cisplatin on Beas-2b human lung epithelial cells using viability assays (clonogenic and XTT) and single cell jel electrophoresis (Comet) assay. In our study to determine cytotoxic effects of quinic acid we performed clonogenic assay and for cisplatin we used XTT formazan dye assay. For cytotoxicity assays, Beas-2b cells exposed to several doses of quinic acid (12 to 18 µM) and cisplatin (0,5 to 50 μM) and survival curves were prepared. IC50 dose of quinic acid was found as 15,48 µM with clonogenic assay. IC50 dose of cisplatin was found as 19 µM with XTT assay. Optimal quinic acid concentrations were chosen as (5 and 10 μM) and these doses were used alone or in combination with (9,5 µM and 19 µM) cisplatin doses. In consequences of comet assay we have been identified increasing genotoxicity in the highest dose of quinic acid at statistically significant levels.With cisplatin treatment at a dose of 9,5 µM the genotoxicity was observed to increase as statistically significant, in the IC50 dose rate of cisplatin we did not see any significant increase in the genotoxicity. In combined dose applications with 9,5 µM cisplatin and 5 and 10 µM quinic acid we found no significant increase or decrease in tail lenght, tail % DNA and olive tail moment parameters. This means that quinic acid was not anti-genotoxic. While high dose cisplatin (19 µM) showed significantly decreased genotoxic effects, 5 µM quinic acid had no effects in combination with this cisplatin dose. However 10 µM quinic acid increased the genotoxic effects of high dose of cisplatin in combination showing the probable apoptosis blocking effect of quinic acid. So we could observe the exact level of damaged cells. Consequently, quinic acid did not show any antigenotoxic effects with cisplatin treatment and it should be applied further apoptosis and genotoxicity tests to better understand the exact mechanism of quinic acid.
This study investigated the cytotoxic and genotoxic effects of quinic acid alone or its anti-genotoxic effects in combination with cisplatin on Beas-2b human lung epithelial cells using viability assays (clonogenic and XTT) and single cell jel electrophoresis (Comet) assay. In our study to determine cytotoxic effects of quinic acid we performed clonogenic assay and for cisplatin we used XTT formazan dye assay. For cytotoxicity assays, Beas-2b cells exposed to several doses of quinic acid (12 to 18 µM) and cisplatin (0,5 to 50 μM) and survival curves were prepared. IC50 dose of quinic acid was found as 15,48 µM with clonogenic assay. IC50 dose of cisplatin was found as 19 µM with XTT assay. Optimal quinic acid concentrations were chosen as (5 and 10 μM) and these doses were used alone or in combination with (9,5 µM and 19 µM) cisplatin doses. In consequences of comet assay we have been identified increasing genotoxicity in the highest dose of quinic acid at statistically significant levels.With cisplatin treatment at a dose of 9,5 µM the genotoxicity was observed to increase as statistically significant, in the IC50 dose rate of cisplatin we did not see any significant increase in the genotoxicity. In combined dose applications with 9,5 µM cisplatin and 5 and 10 µM quinic acid we found no significant increase or decrease in tail lenght, tail % DNA and olive tail moment parameters. This means that quinic acid was not anti-genotoxic. While high dose cisplatin (19 µM) showed significantly decreased genotoxic effects, 5 µM quinic acid had no effects in combination with this cisplatin dose. However 10 µM quinic acid increased the genotoxic effects of high dose of cisplatin in combination showing the probable apoptosis blocking effect of quinic acid. So we could observe the exact level of damaged cells. Consequently, quinic acid did not show any antigenotoxic effects with cisplatin treatment and it should be applied further apoptosis and genotoxicity tests to better understand the exact mechanism of quinic acid.
Description
Keywords
Kinik asit, Cisplatin, Fenolik bileşikler, Komet testi, Sitotoksisite, Quinic acid, Cisplatin, Phenolic compounds, Comet assay, Cytotoxicity
Citation
Kocaoğlu, E. (2014). Kinik asidin sitotoksik/genotoksik ve antigenotoksik etkilerinin sağlıklı akciğer epitel hücre hatlarında belirlenmesi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.