İlaç ve diğer kimyasallara bağlı hücresel toksisite mekanizmalarının araştırılması
Küçük Resim Yok
Dosyalar
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
İlaçların da dahil olduğu kimyasalların kendilerinin ve/veya metabolitlerinin neden olabileceği hücresel toksisite, adaptasyon, koruyucu veya onarım mekanizmalarının yetersiz kalması sonucu doku hatta organizmanın canlılığını tamamen yitirmesine neden olabilir. Bu tez kapsamında CHO-K1 hücrelerinde farklı farmakolojik etkilere sahip sekiz ilacın sitotoksisite profili, MTT yöntemi ile incelenmiştir. Belirgin sitotoksisite gösteren fakat literatürde sitotoksisite mekanizmaları tam olarak açıklanamamış klozapin, diklofenak ve nifedipin ile hem CHO-K1 hem de HepG2 hücrelerinde ileri deneyler gerçekleştirilmiştir. Bu hücrelerde gözlenen sitotoksisitenin reaktif metabolit aracılıklı olup olmadığı, besiyerine “yakalayıcı ajan” nükleofilik ajan olarak glutatyon ve potasyum siyanür eklenmesi ile araştırılmış ve sitotoksisitedeki olası değişim, MTT testi ve LDH sızma testi ile incelenmiştir. Glutatyonun klozapin sitotoksisitesini anlamlı şekilde azaltması, sitotoksisiteden reaktif nitrenyum iyonunun sorumlu olabileceği düşüncesini desteklemiştir. Bu kanıyı güçlendirmek için hücreler klozapin ile glutatyon varlığında inkübe edilmiş ve nitrenyum iyonu-glutatyon konjugatı (KLZ-SG) oluşumu LC-MS/MS yöntemiyle izlenmiştir. Reaktif metabolit oluşumunu katalize eden CYP enzimleri spesifik inhibitörler yardımıyla araştırılmış, ayrıca ilaçların indüklediği sitotoksisite üzerinde inhibitörlerin etkileri incelenmiştir. Sitotoksik mekanizmada yer alması olası bir süreç olarak oksidatif stres, birkaç farklı parametre ile analiz edilmiştir. İlk olarak dokularda olduğu gibi inkübasyon ortamında da bulunması olası geçiş metallerinin CHO-K1 ya da HepG2 hücrelerinde ilaçların indüklediği sitotoksisite ya da membran hasarında rol alıp almadıkları, şelatör ajanlar varlığında her iki toksik sonlanımın analiziyle araştırılmıştır. İkinci olarak sitoplazmada reaktif oksijen türleri varlığında oksitlenerek sinyal veren DCFH varlığında hücre-ilaç inkübasyonları gerçekleştirilmiştir. Oksidatif stresle ilişkili üçüncü ve son test, membran lipitlerinin oksidatif hasarının parçalanma ürünlerinden malondialdehitin (MDA) inkübasyon ortamında ölçülmesi olmuştur. Genel sitotoksisite mekanizmalarından birisi olarak mitokondriyel ATP üretiminin engellenmesi ve mitokondrilerde yapısal hasar, izole mitokondrilerde klozapin, diklofenak ve nifedipin yanında valproik asit ve ilaç dışı madde olarak ATP sentez inhibitörü rotenon, atraktilozid ve Atractylis gummifera bitkisinden saflaştırılan iki fraksiyon varlığında araştırılmış, ATP ölçümüne ek olarak mitokondriyel geçirgenlik dönüşüm porları (MPTP) indüksiyonu test edilmiştir. İzole mitokondri inkübasyonlarında ayrıca mitokondriyel oksidatif stres indüksiyonu, MDA ölçülerek izlenmiştir. İlaçların mitokondriyel toksisiteleri, farklı oranlarda glukoz ve galaktoz içeren besiyerlerinde kültür edilen ve mitokondriyel toksisiteye duyarlı hale getirilen HepG2 hücrelerinde de araştırılmıştır. Son olarak klozapin’in HepG2 hücrelerinden uzaklaştırılmasında ektraselüler veziküllerin rolü araştırılmıştır. Elde edilen sonuçlara göre klozapin’in sitotoksisitesinde CYP aracılıklı oluşan reaktif nitrenyum iyonunun rol oynadığı, membran hasarının temelinde artan reaktif oksijen türleri ve lipit peroksidasyonunun olduğu gösterilmiştir. Ayrıca klozapin sitotoksisitesinde mitokondrinin rol oynamadığı ve klozapin’in hücrelerden atılımının ektraselüler veziküller ile gerçekleşmediği saptanmıştır. Diklofenak’tan oluşabilecek reaktif tiyil yapılarının CHO-K1 hücrelerindeki toksisiteden sorumlu olabileceği gözlenmiştir. Bunun yanında hem izole mitokondrilerde hem de hücrelerde artan reaktif oksijen türleri ve MDA miktarının diklofenak sitotoksisitesinde rol oynadığı tespit edilmiştir. Hücrelerde enzimler aracılıklı reaktif metaboliti hakkında bilgi bulunmayan nifedipin’in sitotoksisitesinde yumuşak ve/veya sert elektrofilik metabolitlerin rol oynayabileceği gözlenmiştir. Diklofenak’ta olduğu gibi nifedipin sitotoksisitesinde de mitokondriyel ve stoplazmik oksidatif stres yer almaktadır.
Cellular toxicity caused by chemicals including drugs and/or their metabolites may result in loss of viability of tissues, even organisms because of insufficiency of cellular adaptative, protective, or repair mechanisms. Within the scope of this thesis, the cytotoxicity profiles of eight drugs with different pharmacological effects were investigated by MTT on CHO-K1 cells. Further experiments were conducted with clozapine, diclofenac, and nifedipine leading significant cytotoxicity in CHO-K1 and HepG2 cells, as their mechanisms of cytotoxicity were not fully explained in the literature. Nucleophilic agents (glutathione and potassium cyanide) were used to investigate whether cytotoxicity observed in these cells were mediated by reactive metabolites as “trapping agents” and the changes in cytotoxicity were monitored by MTT test and LDH leakage assay. Significant decrease in clozapine-induced cytotoxicity after addition of GSH supported the hypothesis that reactive nitrenium ion is responsible for the observed cytotoxicity. To confirm this, cells were incubated with clozapine in the presence of GSH, and nitrenium ion-glutathione conjugate (KLZ-SG) was monitored by LC-MS/MS. Role of cytochrome P450 (CYP) enzymes that catalyse the formation of reactive metabolites was investigated in the presence of specific inhibitors in drug-induced cytotoxicity. Oxidative stress that likely being involved in cytotoxic mechanism was analysed by several parameters. First, any possible role of transition metals in drug-induced cytotoxicity or membrane damage in cells was explored in the presence of chelator agents by analysing both toxic end-points. Second, incubations were performed in the presence of DCFH that gives a fluorescent signal upon oxidation by reactive oxygen species in cytoplasm. The third and the last test on oxidative stress parameters was to measure of malondialdehyde (MDA) level that is one of the oxidative degradation products of membrane lipids. Change in mitochondrial function or structure is one of the general cytotoxicity mechanisms. Hence isolated mitochondria was exposured to clozapine, diclofenac, nifedipine and valproic acid, as well as non-drug rotenone, atractyloside and two purified fractions from Atractylis gummifera and changes in ATP levels together with induction of mitochondrial permeability transition pore (MPTP) formation were measured. In addition, mitochondrial oxidative stress induction was monitored by measuring MDA in isolated mitochondrial incubations. Apart from isolated mitochondria studies, HepG2 cells which were sensitive to mitochondrial toxicans by culturing in different amounth of glucose or galactose were also used for evaluation of mitochondrial toxicity. Finally, role of extracellular vesicules in elimination of clozapine from HepG2 cells was investigated. Accoding our results formation of reactive nitrenium ion, increase of reactive oxygene species and MDA play role in clozapine-induced cytotoxicity and membrane damage. Also, mitochondrial toxicity was not a part of clozapine-induced cytotoxicity and extracellular vesicules did not play role in elimination of clozapine or metabolites. Reactive thiyl radicals formed via further reactions of glutathione conjugates might play role in diclofenac-induced cytotoxicty in CHO-K1 cells. In our studies increase of ROS and MDA levels in both isolated mitochondria and CHO-K1 cells was suggested one of the toxicity mechanisms of diclofenac. Our results showed that nifedipine which has no study about reactive metabolites produced via bioactivation in cell, might lead to cytotoxicity by forming soft and/or hard electrophilic metabolites in CHO-K1 cells. Similar to diclofenac, increase of ROS and MDA formation in isolated mitochondria and CHO-K1 cells took part in nifedipine-induced membrane damage.
Cellular toxicity caused by chemicals including drugs and/or their metabolites may result in loss of viability of tissues, even organisms because of insufficiency of cellular adaptative, protective, or repair mechanisms. Within the scope of this thesis, the cytotoxicity profiles of eight drugs with different pharmacological effects were investigated by MTT on CHO-K1 cells. Further experiments were conducted with clozapine, diclofenac, and nifedipine leading significant cytotoxicity in CHO-K1 and HepG2 cells, as their mechanisms of cytotoxicity were not fully explained in the literature. Nucleophilic agents (glutathione and potassium cyanide) were used to investigate whether cytotoxicity observed in these cells were mediated by reactive metabolites as “trapping agents” and the changes in cytotoxicity were monitored by MTT test and LDH leakage assay. Significant decrease in clozapine-induced cytotoxicity after addition of GSH supported the hypothesis that reactive nitrenium ion is responsible for the observed cytotoxicity. To confirm this, cells were incubated with clozapine in the presence of GSH, and nitrenium ion-glutathione conjugate (KLZ-SG) was monitored by LC-MS/MS. Role of cytochrome P450 (CYP) enzymes that catalyse the formation of reactive metabolites was investigated in the presence of specific inhibitors in drug-induced cytotoxicity. Oxidative stress that likely being involved in cytotoxic mechanism was analysed by several parameters. First, any possible role of transition metals in drug-induced cytotoxicity or membrane damage in cells was explored in the presence of chelator agents by analysing both toxic end-points. Second, incubations were performed in the presence of DCFH that gives a fluorescent signal upon oxidation by reactive oxygen species in cytoplasm. The third and the last test on oxidative stress parameters was to measure of malondialdehyde (MDA) level that is one of the oxidative degradation products of membrane lipids. Change in mitochondrial function or structure is one of the general cytotoxicity mechanisms. Hence isolated mitochondria was exposured to clozapine, diclofenac, nifedipine and valproic acid, as well as non-drug rotenone, atractyloside and two purified fractions from Atractylis gummifera and changes in ATP levels together with induction of mitochondrial permeability transition pore (MPTP) formation were measured. In addition, mitochondrial oxidative stress induction was monitored by measuring MDA in isolated mitochondrial incubations. Apart from isolated mitochondria studies, HepG2 cells which were sensitive to mitochondrial toxicans by culturing in different amounth of glucose or galactose were also used for evaluation of mitochondrial toxicity. Finally, role of extracellular vesicules in elimination of clozapine from HepG2 cells was investigated. Accoding our results formation of reactive nitrenium ion, increase of reactive oxygene species and MDA play role in clozapine-induced cytotoxicity and membrane damage. Also, mitochondrial toxicity was not a part of clozapine-induced cytotoxicity and extracellular vesicules did not play role in elimination of clozapine or metabolites. Reactive thiyl radicals formed via further reactions of glutathione conjugates might play role in diclofenac-induced cytotoxicty in CHO-K1 cells. In our studies increase of ROS and MDA levels in both isolated mitochondria and CHO-K1 cells was suggested one of the toxicity mechanisms of diclofenac. Our results showed that nifedipine which has no study about reactive metabolites produced via bioactivation in cell, might lead to cytotoxicity by forming soft and/or hard electrophilic metabolites in CHO-K1 cells. Similar to diclofenac, increase of ROS and MDA formation in isolated mitochondria and CHO-K1 cells took part in nifedipine-induced membrane damage.
Açıklama
Anahtar Kelimeler
Sitotoksisite, CHO-K1, HepG2, İlaç, Reaktif Metabolit, Oksidatif Stres, Cytotoxicity, CHO-K1, HepG2, Drug, Reactive Metabolite, Oxidative Stress
