Bakır-çinko alaşım nanopartiküllerinin in vitro sitotoksik ve genotoksik etkilerinin araştırılması
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Date
2013
Authors
Kumbıçak, Ümit
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Publisher
Uludağ Üniversitesi
Abstract
Bu çalışmada Cu-Zn alaşım nanopartiküllerin BEAS-2B insan akciğer epitel hücreleri üzerindeki sitotoksik ve genotoksik etkileri araştırılmıştır. Sitotoksik etkilerin belirlenmesi için XTT ve klonojenik test kullanılmıştır. Genotoksik etkiler ise mikronüklus, komet ve ?-H2AX fokus testleri kullanılarak belirlenmiştir. Ayrıca, apoptotik ve nekrotik etkiler ile ROS oluşumunun belirlenmesi için M30, M65 ve ROS testleri uygulanmıştır. Deneylerden önce, kullanılan nanopartikül süspansiyonunun karakterizasyonu için zeta potansiyeli, DLS ve TEM analizleri kullanılarak gerçekleştirilmiştir. XTT ve Klonojenik testler için BEAS-2B hücreleri 24 saat süresince 0,1 ile 100 µg/ml değişen dozlarda Cu-Zn alaşım nanopartiküllerine maruz bırakılmışlardır. Elde edilen IC50 değerine (4,66 µg/ml) dayanarak genotoksisite testlerinde kullanılacak dozlar (0,1-3,2 µg/ml) belirlenmiştir. Çalışılan süspansiyonun karakterizasyonu sonucunda zeta potansiyeli -22 mV ve ortalama partikül büyüklüğü de 200 nm olarak belirlenmiştir. TEM analizlerinde ayrıca Cu-Zn alaşım nanopartiküllerinin sitoplazma içerisinde lokalize olduğu belirlenmiştir. Mikronükleus, komet ve ?-H2AX fokus testlerinin sonucunda Cu-Zn alaşım nanopartiküllerinin kromozomal hasarlar yanında tek ve çift iplik DNA kırıklarına yol açtığı belirlenmiştir. Çalışmamızda belirlenen M30:M65 oranları ise hücre ölümünün ağırlıklı olarak nekroz yoluyla gerçekleştiğini göstermiştir. Ayrıca, elde ettiğimiz bulgular gözlenen sitotoksik ve genotoksik etkilerden hücre içi ROS oluşumunun sorumlu olabileceğini göstermiştir.
In the present study, cytotoxic and genotoxic effect of copper-zinc (Cu-Zn) alloy nanoparticles on BEAS-2B human epithelial cells were evaluated. XTT test and clonogenic assay were used to determine cytotoxic effects. Genotoxic effects were evaluated using micronucleus, comet and -H2AX foci assays. Furthermore, M30, M65 and ROS assays were applied to evaluate apoptotic and necrotic effects as well as intracellular reactive oxygen species formation, respectively. Before the experiments, characterization of the nanoparticle suspension was carried out using zeta potential, DLS and TEM analyses. For XTT and clonogenic assays, BEAS-2B cells were exposed the different concentrations (0,1-100 µg/ml) of Cu-Zn alloy nanoparticles. Based on the determined IC50 value (4,66 µg/ml), concentrations to be used in genotoxicity experiments (0,1-3,2 µg/ml) were determined. Characterization of the studied suspension showed that the zeta potential was 22 mV and the average size was 200 nm. TEM analyses further revealed the intracellular localization of Cu-Zn alloy nanoparticles in cell cytoplasm. Analysis of micronucleus, comet and -H2AX foci frequencies showed that exposure to Cu-Zn alloy nanoparticles induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Evaluation of M30:M65 ratios suggest that cell death was predominantly due to necrosis. Our results further indicated that increased intracellular ROS formation could be responsible from the observed cytotoxic and genotoxic effects.
In the present study, cytotoxic and genotoxic effect of copper-zinc (Cu-Zn) alloy nanoparticles on BEAS-2B human epithelial cells were evaluated. XTT test and clonogenic assay were used to determine cytotoxic effects. Genotoxic effects were evaluated using micronucleus, comet and -H2AX foci assays. Furthermore, M30, M65 and ROS assays were applied to evaluate apoptotic and necrotic effects as well as intracellular reactive oxygen species formation, respectively. Before the experiments, characterization of the nanoparticle suspension was carried out using zeta potential, DLS and TEM analyses. For XTT and clonogenic assays, BEAS-2B cells were exposed the different concentrations (0,1-100 µg/ml) of Cu-Zn alloy nanoparticles. Based on the determined IC50 value (4,66 µg/ml), concentrations to be used in genotoxicity experiments (0,1-3,2 µg/ml) were determined. Characterization of the studied suspension showed that the zeta potential was 22 mV and the average size was 200 nm. TEM analyses further revealed the intracellular localization of Cu-Zn alloy nanoparticles in cell cytoplasm. Analysis of micronucleus, comet and -H2AX foci frequencies showed that exposure to Cu-Zn alloy nanoparticles induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Evaluation of M30:M65 ratios suggest that cell death was predominantly due to necrosis. Our results further indicated that increased intracellular ROS formation could be responsible from the observed cytotoxic and genotoxic effects.
Description
Keywords
Nanotoksikoloji, Bakır-çinko alaşım nanopartikülleri, BEAS- 2B hücreler, Sitotoksisite, Genotoksisite, Nanotoxicology, Copper-zinc alloy nanoparticles, BEAS-2B cells, Cytotoxicity, Genotoxicity
Citation
Kumbıçak, Ü. (2018). Bakır-çinko alaşım nanopartiküllerinin in vitro sitotoksik ve genotoksik etkilerinin araştırılması. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.