Bitkisel fenolik bir bileşik olan klorojenik asidin radyoduyarlılık oluşturma etkisinin in vitro genotoksisite ve enzim test sistemleri yardımıyla araştırılması
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Date
2013
Authors
Teksoy, Özgün
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Publisher
Uludağ Üniversitesi
Abstract
Yapılan bu çalışmada klorojenik asidin radyasyona maruz bırakılan Caco2 insan kolon adenokarsinom hücreleri üzerindeki radyoduyarlılık etkileri tek hücre jel elektroforezi (Komet) ve M30 testi kullanılarak araştırılmıştır. Çalışmamızda, klorojenik asit ve radyasyonun tek başlarına oluşturdukları sitotoksik etkilerin belirlenmesi amacıyla XTT ve klonojenik test uygulanmıştır. Çeşitli dozlarda klorojenik asit ( 11?5780 µM) ve radyasyona (0,5 ? 6 Gy) maruz bırakılan Caco2 hücrelerinde hayatta kalış eğrileri hazırlanmıştır. XTT testi ile klorojenik asidin IC50 dozu 1816 µM olarak belirlenmiştir. Klonojenik test sonuçlarına göre ise çalışmamızda kullanılacak radyasyon dozu 2 Gy olarak tespit edilmiştir. Komet ve M30 apoptoz testi için kullanılacak optimum klorojenik asit konsantrasyonları (908, 1816 ve 2724 µM) ve radyasyon dozu (2 Gy) olarak belirlenmiştir. Bu konsantrasyonlar tek başına ve kombine olarak kullanılmıştır. Komet testi sonucunda klorojenik asitin belirlenen konsantrasyonlarda artan bir genotoksisiteye neden olduğu tespit edilmiştir. Radyasyon ile muamelede ise 2 Gy dozda genotoksisitede istatistiksel anlamlı şekilde artış olduğu gözlenmiştir. Radyasyon hassasiyetini belirlemek için gerçekleştirilen kombine doz uygulamalarında ise, hücreler 2 Gy radyasyona maruz bırakılmadan 24 saat önce 908, 1816 ve 2724 µM konsantrasyonlardaki klorojenik asit ile muamele edilmişlerdir. Radyasyona maruz bırakılmadan önce klorojenik asit uygulanması durumunda Caco2 hücrelerinde DNA hasarının tek başına radyasyon verilen hücrelere göre istatistiksel anlamlı arttığı gösterilmiştir. M30 apoptoz testinde ise tek başına radyasyon ile muamele edilen hücrelerle karşılaştırıldığında klorojenik asidin apoptoz oranını istatistiksel anlamlı oranda arttırmadığı belirlenmiştir. Elde ettiğimiz sonuçlara göre, radyasyon ile kombine uygulanan klorojenik asidin IC 50 konsantrasyonu üzerindeki dozlarda radyasyonca indüklenen sitotoksik ve genotoksik etkiyi arttırdığı ancak apoptoz oranını arttırmadığı yani Caco2 kanser hücre hatlarında radyoduyarlılık etkisinin olmadığı ortaya konmuştur.
This study investigated the radiosensitivity effects of chlorogenic acid alone or in combination with radiation on Caco2 human colon cancer cells using viability assays (clonogenic and XTT), single cell jel electrophoresis (Comet) and M30 apoptosis enzyme assays. The aim of our work, to determine firstly cytotoxic effects of chlorogenic acid and radiation alone or in combination using XTT and clonogenic assay. Survival curve in Caco2 cells exposed to several doses of chlorogenic acid (11-5780µM) and radiation (0.5-6 Gy) was prepared. IC50 dose of chlorogenic acid was found as 1816 vµM with XTT assay. According to results of clonogenic assay, optimum radiation dose to be used in our study was determined as 2 Gy. Optimal chlorogenic acid concentrations were chosed as IC25, IC50 and IC75 doses (908, 1816 and 2724 µM respectively) and these doses were used alone and in combination with 2 Gy radiation dose. In consequence of comet assay we have been identified increasing genotoxicity in different concentrations of chlorogenic acid at statistically significant levels. There was also significant increase in DNA damage ratios at 2 Gy radiation dose. We combined three doses of chlorogenic acid with a single radiation dose in comet assay and found that while chlorogenic acid may have ability to induce DNA damage alone, combination with radiation did not show increased DNA damage compared with single radiation treatment. The chlorogenic acid showed radioprotective effect at the lower doses. We used M30 Elisa apoptosis assay to understand the radiosensitivity effect of chlorogenic acid on radiation induced apoptosis on Caco2 cells. Single radiation dose increased the frequency of cytokeratin 18 M30 antibodies compared to solvent control. However chlorogenic acid combined with radiation did not increase the M30 antibody level and did not affect apoptosis induced by 2 Gy radiation in Caco2 cell lines. Consequently chlorogenic acid increased the cytotoxic and genotoxic effects induced by single dose of radiation at the higher doses such as IC50 and IC75 but it did not elevate the apoptotic effect of single radiation treatment in Caco2 human colon adenocarsinoma cells.
This study investigated the radiosensitivity effects of chlorogenic acid alone or in combination with radiation on Caco2 human colon cancer cells using viability assays (clonogenic and XTT), single cell jel electrophoresis (Comet) and M30 apoptosis enzyme assays. The aim of our work, to determine firstly cytotoxic effects of chlorogenic acid and radiation alone or in combination using XTT and clonogenic assay. Survival curve in Caco2 cells exposed to several doses of chlorogenic acid (11-5780µM) and radiation (0.5-6 Gy) was prepared. IC50 dose of chlorogenic acid was found as 1816 vµM with XTT assay. According to results of clonogenic assay, optimum radiation dose to be used in our study was determined as 2 Gy. Optimal chlorogenic acid concentrations were chosed as IC25, IC50 and IC75 doses (908, 1816 and 2724 µM respectively) and these doses were used alone and in combination with 2 Gy radiation dose. In consequence of comet assay we have been identified increasing genotoxicity in different concentrations of chlorogenic acid at statistically significant levels. There was also significant increase in DNA damage ratios at 2 Gy radiation dose. We combined three doses of chlorogenic acid with a single radiation dose in comet assay and found that while chlorogenic acid may have ability to induce DNA damage alone, combination with radiation did not show increased DNA damage compared with single radiation treatment. The chlorogenic acid showed radioprotective effect at the lower doses. We used M30 Elisa apoptosis assay to understand the radiosensitivity effect of chlorogenic acid on radiation induced apoptosis on Caco2 cells. Single radiation dose increased the frequency of cytokeratin 18 M30 antibodies compared to solvent control. However chlorogenic acid combined with radiation did not increase the M30 antibody level and did not affect apoptosis induced by 2 Gy radiation in Caco2 cell lines. Consequently chlorogenic acid increased the cytotoxic and genotoxic effects induced by single dose of radiation at the higher doses such as IC50 and IC75 but it did not elevate the apoptotic effect of single radiation treatment in Caco2 human colon adenocarsinoma cells.
Description
Keywords
Klorojenik asit, İyonize radyasyon, Radyoduyarlılık, Caco2 kolon adenokarsinom hücreleri, Apoptoz, DNA hasarı, Komet, XTT canlılık testi, Chlorogenic acid, Ionizing radiation, Radiosensitivity, Caco2 colon adenocarcinoma cells, Apoptosis, DNA damage, Comet, XTT viability assay
Citation
Teksoy, Ö. (2013). Bitkisel fenolik bir bileşik olan klorojenik asidin radyoduyarlılık oluşturma etkisinin in vitro genotoksisite ve enzim test sistemleri yardımıyla araştırılması. Yayınlanmamış yüksek lisans tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.