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Öğe Bioactivation of clozapine by mitochondria of the murine heart: Possible cause of cardiotoxicity(Elsevier Ireland Ltd, 2021) Arzuk, Ege; Karakus, Fuat; Orhan, HilmiThe mechanism of clozapine-associated cardiotoxicity has not been elucidated. The formation of a reactive nitrenium ion from the drug has been suggested as the cause, however, the reason why the heart is a target remains unknown. The heart is one of the most perfused organs; therefore, it contains a large number of mitochondria per cell; these organelles are responsible for both oxygen metabolism and energy production due to high energy expenditure. Given that mitochondria play critical roles in cellular homeostasis and maintenance, this study tested the hypothesis that cardiac mitochondria are both a target and initiator of clozapine-induced cardiotoxicity through activating the drug. We investigated whether murine heart receives a relatively high amount of systemically administered drug (20 mg/kg, i.p., Wistar albino rats) and whether cardiac mice (Swiss albino) and rat (Wistar albino) mitochondria locally activate clozapine (100 mu M) to a reactive metabolite. We observed a relatively large distribution of clozapine to heart tissue as well as the formation of reactive metabolites by cardiac mitochondria in situ. Mitochondrial cytochrome P450 enzymes (CYP) in cardiac tissue responsible for biotransformation of clozapine were also characterized. CYP3A4 has been found to be the major enzyme catalyzes CLZ bioactivation, while CYP1A largely and CYP3A4 partially catalyzes the formation of stable metabolites of CLZ. At 100 mu M concentration, clozapine caused a significant decline in mitochondrial oxygen consumption rate in vitro as much as positive control (antimycin A), while it did not induce mitochondrial permeability transition pore opening. These data provide an explanation as to why the heart is a target for clozapine adverse effects.Öğe Celecoxib inhibits NLRP1 inflammasome pathway in MDA-MB-231 Cells(Springer, 2024) Arzuk, Ege; Birim, Dervis; Armagan, GulizNLRP1 is predominantly overexpressed in breast cancer tissue, and the evaluated activation of NLRP1 inflammasome is associated with tumor growth, angiogenesis, and metastasis. Therefore, targeting NLRP1 activation could be a crucial strategy in anticancer therapy. In this study, we investigated the hypothesis that NLRP1 pathway may contribute to the cytotoxic effects of celecoxib and nimesulide in MDA-MB-231 cells. First of all, IC50 values and inhibitory effects on the colony-forming ability of drugs were evaluated in cells. Then, the alterations in the expression levels of NLRP1 inflammasome components induced by drugs were investigated. Subsequently, the release of inflammatory cytokine IL-1 beta and the activity of caspase-1 in drug-treated cells were measured. According to our results, celecoxib and nimesulide selectively inhibited the viability of MDA-MB-231 cells. These drugs remarkably inhibited the colony-forming ability of cells. The expression levels of NLRP1 inflammasome components decreased in celecoxib-treated cells, accompanied by decreased caspase-1 activity and IL-1 beta release. In contrast, nimesulide treatment led to the upregulation of the related protein expressions with unchanged caspase-1 activity and increased IL-1 beta secretion. Our results indicated that the NLRP1 inflammasome pathway might contribute to the antiproliferative effects of celecoxib in MDA-MB-231 cells but is not a crucial mechanism for nimesulide.Öğe Celecoxib inhibits NLRP1 inflammasome pathway in MDA-MB-231 Cells (JUL, 10.1007/s00210-024-03286-2, 2024)(Springer, 2024) Arzuk, Ege; Birim, Dervis; Armagan, Guliz[Abstarct Not Available]Öğe Design, synthesis, and antiproliferative activities of novel thiazolyl-pyrazole hybrid derivatives(Springer Birkhauser, 2023) Kuzu, Burak; Erguc, Ali; Karakus, Fuat; Arzuk, EgeIn this study, a series of derivatives of thiazolyl-pyrazole hybrid structures were designed to search for new heterocyclic compound-based antitumor agents. The designed target structures were synthesized with easy, practical, and efficient procedures. The antiproliferative effect of the synthesized compounds against cancer cell lines A549, MCF-7, and HepG2 was evaluated regarding inhibition concentration and selectivity index against healthy cell line CCD-34Lu. The results overall showed that the compounds had high antiproliferation against cancer cells compared to the doxorubicin-positive control. In particular, compound 11 A549 (SI: 3.58) and HepG2 (SI: 12.36) had high selectivity in cancer cell lines, while compounds 10h and 10o had high selectivity (SI: 10.74 for both) in MCF-7 cancer cell lines. The calculated theoretical pharmacokinetic properties revealed that they could be suitable drug candidates. In addition, in vitro test results indicate a correlation between the structure-activity relationships of the compounds. The various molecular modifications of thiazolyl-pyrazole hybrid compounds are promising for developing new anticancer drug candidates.Öğe Discovery of New Pyrazole-Tosylamide Derivatives as Apoptosis Inducers Through BCL-2 Inhibition and Caspase-3 Activation(Wiley - VCH Verlag Gmbh, 2024) Kuzu, Burak; Arzuk, EgeIn this presented study, a series of new carbonitrile-substituted pyrazole-tosyl amide derivatives were designed and synthesized according to previous studies. The antiproliferative effects of the synthesized compounds on MDA-MB-231, MCF-7, HepG2, PC-3, and A549 cancer cell lines were assessed by MTT assay compared with non-cancerous cells. The results demonstrate that compounds 9d, 9e, and 9f had a higher antiproliferative effect (IC50 <10 mu M) against both breast cancer cells. To investigate the ability of these compounds (9d-f) to induce apoptosis against breast cancer cells, BCL-2 levels and Caspase-3 activities of compound-treated breast cancer cell lines were measured by ELISA. The results revealed that these compounds significantly inhibited the levels of anti-apoptotic protein BCL-2 and increased the activity of apoptotic protein Caspase-3 in MDA-MB-231 and MCF-7 cells. Molecular docking studies confirmed that the selected compounds have high binding affinity towards the active site in the crystal structures of BCL-2 and Caspase-3. Moreover, drug-likeness and pre-ADMET evaluation showed that the compounds had suitable drug properties. This study may be a new milestone in terms of the promising importance of carbonitrile-substituted pyrazole-tosyl amide scaffolds as apoptosis-inducing agents for cancer therapy in the future.Öğe İlaç toksisitesinde mitokondrinin rolünün değerlendirilmesi(Ege Üniversitesi, Sağlık Bilimleri Enstitüsü, 2018) Arzuk, Ege; Orhan, HilmiMitokondri hücresel enerjinin üretildiği, oksidatif solunumun ve diğer birçok yaşamsal öneme sahip olayın gerçekleştiği organeldir. Bu nedenle ilaçların neden olduğu mitokondriyel yapısal ya da işlevsel bozukluklar, organizma açısından ciddi sonuçlar doğurabilmektedir. Bu tez kapsamında toksisite mekanizması tam olarak anlaşılamamış olan bazı ilaçların mitokondri üzerindeki etkilerinin araştırılması amaçlanmıştır. Klozapin kullanan hastalarda gözlenen kardiyotoksisite ve parasetamol kullanan hastalarda oluşan nefrotoksisitenin mekanizması henüz anlaşılamamıştır, ancak biyotransformasyonuyla ilişkili olduğu düşünülmektedir. Advers ilaç reaksiyonlarının önemli bir kısmının ilaç molekülünden oluşan reaktif metabolitlerle ilişkili olduğu bilinmektedir. Bu nedenle her iki ilacın toksisitesinde mitokondriyel ve mikrozomal biyoaktivasyonun rolü araştırılmıştır. Bu amaçla öncelikle hastalardan elde edilen karaciğer ve böbrek dokularından hazırlanan mikrozomlar parasetamolle inkübe edilmiş, oluşan reaktif metabolit miktarları ve biyoaktivasyonu katalize eden enzim aktivitelerinde bireyler arası ve organlar arası farklılıklar ortaya konmuştur. Çalışmanın hipotezlerinden birisi olan "ilaçların mitokondri içerisinde lokal reaktif metabolit üreterek toksisiteye yol açması" olasılığı parasetamol ve klozapin için test edilmiş, bu amaçla öncelikle çeşitli tekniklerle saf mitokondriyel fraksiyonlar elde edilmiş ve saflık Western blot tekniği ile gösterilmiştir. Klozapinin fare karaciğer ve kalp mitokondriyel fraksiyonları ile inkübasyonları gerçekleştirilmiş, mitokondriyel enzimler aracılığıyla stabil metabolitlerin yanı sıra reaktif metabolit oluşumu gösterilmiştir. Benzer şekilde mitokondriler tarafından parasetamolden reaktif metabolit oluşumu da gösterilmiştir. Mitokondrilerin yapısal ve işlevsel bütünlüğünün göstergesi olarak "mitochondrial permeability transition pore (MPTP)" oluşumu ölçülmüş ve 3 ay süreyle doku ya da izole halde -86oC'de saklanan mitokondrilerin bütünlüğünü koruduğu gözlenmiştir. Klozapin ve parasetamol dışında olanzapin, nifedipin, diklofenak, valproik asit, metformin, dapson, tiyaprofenik asit ve sülfafenazolün de kalsiyum varlığında ve yokluğunda mitokondrideki MPTP indükleyici etkileri araştırılmış, diklofenak ve valproik asitin kalsiyum tarafından indüklenen MPTP oluşumunu şiddetlendirdiği, ancak hiçbir ilacın tek başına MPTP oluşturmadığı gözlenmiştir. Genlerin aktivitelerini belirleyen önemli olaylardan birisi olan DNA metilasyonunun mitokondriyel DNA'da çalışılan ilaçlar tarafından değiştirilip değiştirilmediği araştırılmış, tüm incelenen ilaçların anlamlı derecede hipometilasyona yol açtığı, klozapinin bu etkisinin reaktif metaboliti aracılığı ile olduğu gösterilmiştir. Araştırılan ilaçların mitokondrilerde gerçekleşen en önemli metabolik süreç olan oksijen tüketim hızını etkileyip etkilemediği incelenmiş, klozapin, olanzapin, nifedipin, valproik asit, dapson ve tiyaprofenik asitin tüketim hızını düşürdüğü, sülfafenazolün ise ilginç bir şekilde artırdığı gözlenmiştir. Son olarak mitokondriyel ilaç biyotransformasyonu açısından önemli olan insan karaciğer ve kalp mitokondrilerinin antioksidan ve konjugasyon enzim aktiviteleri, otopsi sırasında farklı bireylerden elde edilen dokularda incelenmiş, her iki organ mitokondrilerinde GSTT, GSTM, CAT ve Se-GPx aktiviteleri insanda gösterilmiştir. Tez çalışmasında çalışılan ilaçların mitokondride yapısal ve/veya işlevsel etkilerinin incelenmesiyle sadece toksik değil, olası terapötik hedefler için de veri sağlanmış, klozapin-aracılıklı kardiyotoksisite ve parasetamol-aracılıklı nefrotoksisite mekanizmaları açısından yeni fikirler ortaya konmuştur. Geliştirilen bu deneysel yaklaşımlar farklı ilaç, ilaç adayları ya da diğer kimyasal maddelerin olası mitokondriyel toksisitelerini taramak üzere sonraki çalışmalar için yol gösterici olacaktır.Öğe In silico and in vitro evaluation of oxypeucedanin-induced anticancer activity: Mitotoxicity?(2023) Ergüç, Ali; Okur, Hayati; Karakuş, Fuat; Albayrak, Gökay; Arzuk, Ege; Baykan, ŞüraAim: This study aims to evaluate the alterations in Oxypeucedanin (OXY)-mediated anticancer activity in different media. Second aim is to predict the affinity of OXY to electron transfer chain (ETC) complexes. Materials and Methods: MTT and LDH leakage assays were performed with OXY. Molecular docking studies were also conducted to predict the affinity of OXY to ETC complexes. Results: 250 µM OXY reduced viability in glucose media. ?50 µM OXY decreased viability in galactose media. ?50 µM OXY increased membrane disruption in galactose media. Molecular docking studies also showed that OXY might possess the capacity to bind to the inhibition sites of Complex I and IV. Conclusion: Galactose-conditioned media exacerbated the OXY-mediated cytotoxicity. Preliminary results suggested that mitotoxicity might take part in anticancer activity. Furthermore, OXY might cause ETC dysfunctions due to selective inhibition of Complex I and IV.Öğe Inter-individual and inter-organ variability in the bioactivation of paracetamol by human liver and kidney tissues(Elsevier Science Bv, 2018) Arzuk, Ege; Turna, Burak; Sozbilen, Murat; Orhan, HilmiParacetamol (PAR) overdose is associated with massive hepatic injury; it may induce kidney toxicity as well. It is essential to measure organ-specific activities of related CYPs for evaluating the overdose cases. Available HPLC-based methods require high amounts of tissue samples. In order to develop liquid chromatography mass spectrometry (LC-MS)-based methods to process small amounts of human tissues, liver and kidney samples were obtained. Individual microsomes were prepared and incubated with PAR (for quantifying bioactivation), with nifedipine (for measuring CYP3A4 activity) and with p-nitrophenol (for measuring CYP2E1 activity). The small amount of tissue microsomes was sufficient to measure both the formation of NAPQI and the activities of CYP enzymes. Although the sample size in group was relatively low, both NAPQI formation and activity of CYP2E1 were significantly higher in males compared to females in kidney. Considerable variations in the metabolic capacity of individuals were observed for both organs.Öğe Interindividual variability in generating acetaminophen reactive metabolite NAPQI by various human liver and kidney microsomes(Elsevier Ireland Ltd, 2013) Arzuk, Ege; Turna, Burak; Sozbilen, Murat; Orhan, HilmiÖğe Measurement of 8-oxo-7,8-dihydro-2 '-deoxyguanosine in urine by an improved ELISA(Elsevier Ireland Ltd, 2017) Rossner, Pavel, Jr.; Orhan, Hilmi; Koppen, Gudrun; Sakai, Kazuo; Santella, Regina M.; Ambroz, Antonin; Rossnerova, Andrea; Sram, Radim J.; Ciganek, Miroslav; Neca, Jiri; Arzuk, Ege; Mutlu, Neliye; Cooke, Marcus S.Öğe Meme kanseri hücre hattında COX-2 inhibitörü ilaçların inflamazom aktivasyonu üzerine etkisinin araştırılması(Ege Üniversitesi, 2023) Arzuk, Ege; Birim, DervişKronik inflamasyonun meme kanseri prognozunda rolü olduğunu düşünülmektedir. Tümör büyümesini ve maligniteyi arttırıcı etkisi nedeniyle inflamasyon, kanserin önlenmesi ve tedavisi için umut verici bir hedef haline gelmiştir. İnflamasyonun başlangıcında ve devamlılığında bir dizi mekanizma rol oynar. Pirin alanı içeren NOD benzeri reseptör 1 (NLRP1) inflamazomu, kaspaz-1 yoluyla IL-1? gibi inflamatuvar sitokinlerin salınmasından ve inflamasyonun başlatılmasından sorumlu olan oligomerik protein kompleksidir. Siklooksijenaz?2 (COX?2), inflamatuvar uyaranlarla indüklenen ve inflamasyonla ilişkili hastalıkların patogenezine katkıda bulunan bir enzimdir. COX-2 ve/veya NLRP1 inflamazomu ile meme kanseri insidansı, tümör büyüklüğü ve metastazı arasında pozitif bir ilişki olduğu gösterilmiştir. Ancak, COX?2'nin NLRP1 regülasyonundaki rolü kesin değildir, bilgiler sınırlıdır. Projemizde, COX-2 inhibitörleri selekoksib ve nimesulidin meme kanserindeki terapötik etki mekanizması, NLRP1 inflamazom aktivasyonu aracılığıyla araştırılmıştır. Selekoksib ve nimesulid, MDA-MB-231 hücrelerinin canlılığını önemli derecede azaltmış ve hücrelerin koloni oluşturma potansiyeli ve göçünü doza bağlı bir şekilde inhibe etmiştir. Ayrıca, 2 ilaç da proinflamatuvar sitokin IL-1? salgılanmasında ve kaspaz-1 aktivitesinde belirgin bir azalmaya neden olmuştur. Selekoksib, NLRP1 protein miktarını ve NLRP1 inflamazom kaskadında bulunan kaspaz-1 protein miktarını azaltmıştır. Gasdermin ve kaspaz-5 proteinlerinde ise aynı etki gözlenmemiştir. Selekoksibin sitotoksik etkisinde NLRP1 aktivasyon inhibisyonunun önemli olduğunu düşünmekteyiz. Nimesulidin ise NLRP1 inflamazom aktivasyonunda yer alan protein bileşenlerine inhibisyon etkisi yoktur. Ancak, efektör proteinler olan kaspaz-1 ve IL-1? aktivite ve salınımlarındaki azalma olası sitotoksik etkinin bu 2 bileşenin de yer aldığı farklı bir yolak aracılıklı olabileceğini göstermektedir. Bu proje, klinikte kullanılan COX?2 inhibitörlerinin NLRP1 aktivasyonunu nasıl düzenlediğine ve NLRP1'in meme kanserindeki rolüne dair yeni bilgiler sağlanmıştır.;Meme kanseri, COX-2 inhibitörleri, inflamasyon, NLRP1 inflamazom.;Breast cancer, COX-2 inhibitors, inflammation, NLRP1 inflammasome.Öğe Mitochondrial Impact of Organophosphate Pesticide-Induced Cardiotoxicity: An In Silico and In Vitro Study(Sage Publications Inc, 2024) Karakus, Fuat; Arzuk, Ege; Erguc, AliOrganophosphate pesticides are widely used; however, their use is limited due to neurotoxicity and, to a lesser extent, cardiotoxicity in humans. Given the high energy demands of cardiac muscle, which is characterized by a dense population of mitochondria, any damage to these organelles can exacerbate cardiotoxicity. This study aims to elucidate whether the cardiotoxic effects of organophosphate pesticides originate from mitochondrial dysfunction. To investigate this, in silico toxicogenomic analyses were performed using various tools, such as the Comparative Toxicogenomic Database, GeneMANIA, STRING, and Cytoscape. Results revealed that 11 out of the 13 WHO-recommended Class Ia organophosphate pesticides target genes associated with cardiotoxicity. Notably, three of these genes were mitochondrial, with catalase (CAT) being the common differentially expressed gene among parathion, methyl parathion, and phorate. Furthermore, protein-protein interaction analysis indicated a strong association between CAT and superoxide dismutase 2, mitochondrial (SOD2). Subsequently, isolated heart mitochondria were utilized to assess CAT and superoxide dismutase (SOD) activities in vitro. The findings demonstrated that at a concentration of 7.5 ng/mu L, both methyl parathion and phorate significantly decreased CAT activity by approximately 35%. Moreover, phorate reduced total SOD and SOD2 activities by 17% and 19%, respectively, at the same concentration. In contrast, none of the three organophosphate pesticides induced the opening of the mitochondrial permeability transition pore. These results suggest that the reduction in CAT and SOD2 activities, critical antioxidant enzymes, leads to the accumulation of reactive oxygen species within mitochondria, ultimately resulting in mitochondrial damage. This mechanism likely underlies the observed cardiotoxicity induced by these organophosphate pesticides.Öğe Mitochondrial toxicity of selected natural compounds: in vitro assessment and in silico molecular docking and dynamics simulation(Taylor & Francis Ltd, 2024) Erguc, Ali; Albayrak, Gokay; Muhammed, Muhammed Tilahun; Karakus, Fuat; Arzuk, EgePrangos uechtritzii Boiss & Hausskn stands out for its rich bioactive constituents including prantschimgin (PRA), imperatorin (IMP), suberosin (SUB), adicardin (ADI), and oxypeucedanin hydrate (OPH) in the Apiaceae family. Although these molecules contribute to several biological activities, their mitochondrial toxicity were not illuminated in depth with the appropriate in vitro and in silico models. Cell viability studies investigated the cytotoxic activities of molecules in HepG2 cells by replacing glucose with galactose due to Warburg effects. Mitochondrial toxicity (mitotoxicity) parameters such as cellular adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) levels were assessed with cytotoxic concentrations of selected molecules. Molecular docking and dynamics studies were also conducted against mitochondrial electron transport chain (ETC) complexes (I-V) with selected compounds. In vitro results showed that PRA, SUB, and IMP reduced cell viability more in galactose media compared to high glucose media in a dose-dependent manner. PRA, IMP, and SUB decreased ATP levels and MMP, especially in the galactose medium. The in silico study revealed that PRA, IMP, and SUB might bind to complexes I-V at different levels. The docking study demonstrated that PRA had the highest binding potential with the complexes, higher than the standard ligands in some cases. The molecular dynamics (MD) simulation study showed that PRA formed stable complexes with complexes II, III, and IV. In addition, PRA was anticipated to remain inside the binding site of complex II most stably during the 230 ns simulation period. Our study suggests that PRA, IMP, and SUB exhibit mitotoxicity.Öğe Mitochondrial versus microsomal bioactivation of paracetamol by human liver and kidney tissues(Elsevier Ireland Ltd, 2022) Arzuk, Ege; Tokdemir, Mehmet; Orhan, HilmiMitochondria appeared to be a major target for paracetamol (PAR)-induced hepatotoxicity. Studies suggested that microsomal CYPs catalyse bioactivation of PAR to N-acetyl-p-benzoquinone imine (NAPQI), which alkylates mitochondrial proteins, and causes transmission of death signal from mitochondria to nucleus. We hypothesised that local formation of NAPQI within mitochondria seems more likely compared to the translocation of NAPQI. We therefore tested whether the formation of NAPQI may be catalysed by mitochondrial CYPs. Cellular fractions were isolated from human liver and kidney to compare the metabolic capacities. Liver and kidney mitochondria are capable to generate NAPQI. Mitochondrial CYP2E1 and CYP3A4 activities were comparable to the microsomal counterparts in both organs. Previously reported higher kidney microsomal CYP2E1 activity in men compared women were observed in mitochondrial CYP2E1 as well in the present study. On the other hand, no correlation between kidney CYP2E1 activity and quantity of NAPQI formation, as well as no induction on mitochondrial permeability transition pore (mPTP) opening by PAR in kidney mitochondria strongly suggested a different toxicity mechanism in this organ.Öğe Novel Pyrazole Derivatives Bearing Carbonitrile and Substituted Thiazole Moiety for Selective COX-2 Inhibition(Wiley-V C H Verlag Gmbh, 2024) Arzuk, Ege; Karakus, Fuat; Erguc, Ali; Kuzu, BurakIn this study, a series of derivatives of pyrazole hybrid structures containing carbonitrile and substituted thiazole moiety were designed to search for selective COX-2 inhibition. The designed target structures were synthesized with easy, practical, and efficient procedures. COX-1/2 inhibition and cytotoxic effects of the synthesized compounds were evaluated in NIH/3T3 and MDA-MD-231 cell lines for inhibition concentration and selectivity index. The results showed that the compounds have an inhibitory effect with higher selectivity towards COX-2 overall in both cell lines and moderate antiproliferative activity by targeting the breast cancer cell line MDA-MB-231. Among the 19 compounds synthesized (19 a-t), especially compound 19 m was found to be highly effective with COX-2 inhibition of 5.63 mu M in the NIH/3T3 cell line and 4.12 mu M in the MDA-MB-231 cell line. Moreover, molecular docking studies showed that the compounds indeed exhibited higher affinity for the COX-2 active site. The theoretical ADMET properties of the presented compounds were calculated, and the results showed that the compounds may have a more favorable pharmacokinetic effect profile than the selective COX-2 inhibitor Celecoxib, thus promising COX-2 inhibitor drug candidates for the future. A series of derivatives of pyrazole hybrid structures were designed to search for selective COX-2 inhibition. COX-1/2 inhibition and cytotoxic effects of the synthesized compounds were evaluated in NIH/3T3 and MDA-MD-231 cell lines. Moreover, molecular docking, SAR, and ADMET studies showed that the compounds may have a more favorable pharmacokinetic profile, thus promising COX-2 inhibitor drug candidates for the future.imageÖğe Radiolabeled Trastuzumab Solid Lipid Nanoparticles for Breast Cancer Cell: in Vitro and in Vivo Studies(Amer Chemical Soc, 2022) Ozgenc, Emre; Karpuz, Merve; Arzuk, Ege; Gonzalez-Alvarez, Marta; Sanz, Marival Bermejo; Gundogdu, Evren; Gonzalez-Alvarez, IsabelRadiolabeled trastuzumab (TRZ) loaded solid lipid nano-particles (SLNs) were prepared by high shear homogenization and sonication techniques. The apoptosis mechanism of TRZ-SLNs was studied only with the MCF-7 cell line, while the cytotoxicity and cell binding capacity were investigated using breast cancer cells (MCF-7 and MDA-MB-231) and the human keratinocyte cell line (HaCaT). The particle sizes of TRZ-SLNs were found to be below 100 nm, and they possessed a negative charge. The high radiolabeling efficiency and good radiolabeling stability in saline and a cell culture medium were obtained in the results of radiolabeling studies. According to the in vitro studies, TRZ-SLNs were found to be biocompatible, and they effectively induced apoptosis in MCF-7 cells. After the parenteral injection of TRZ-SLNs into rats, a sustained release profile in blood circulation was achieved compared with free drug solution by the evaluation of pharmacokinetic parameters. As a conclusion, the study reveals that Technetium-99m (Tc-99m radiolabeled) TRZ loaded SLN formulations could be promising theranostic agents based on their characterization profiles, in vitro cellular uptake and apoptosis induction capacity, and in vivo pharmacokinetic profiles.Öğe Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells in vitro(Bentham Science Publ Ltd, 2023) Erguec, Ali; Karakus, Fuat; Arzuk, Ege; Mutlu, Neliye; Orhan, HilmiBackground CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. Aim To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. Objective Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. Methods Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYP-catalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. Results The presence of an individual or combined nucleophilic agents significantly diminished CLZ- and NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazole significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DIC-induced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. Conclusion Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.Öğe Urinary 8-oxo-7,8-dihydro-2 '-deoxyguanosine analysis by an improved ELISA: An inter-laboratory comparison study(Elsevier Science Inc, 2016) Rossner, Pavel, Jr.; Orhan, Hilmi; Koppen, Gudrun; Sakai, Kazuo; Santella, Regina M.; Ambroz, Antonin; Rossnerova, Andrea; Sram, Radim J.; Ciganek, Miroslav; Neca, Jiri; Arzuk, Ege; Mutlu, Neliye; Cooke, Marcus S.ELISA is commonly used for the detection of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of whole body oxidative stress. However, the method has been criticized for high inter-laboratory variability and poor agreement with chromatographic techniques. We performed an inter-laboratory comparison of 8-oxodG assessed in 30 urine samples and a urine spiked with four different concentrations of 8-oxodG by ELISA using standardized experimental conditions, including: sample pretreatment with solid-phase extraction (SPE), performing analysis using a commercial kit from a single manufacturer and strict temperature control during the assay. We further compared the ELISA results with high-performance liquid chromatography-tandem mass spectrometry (HPLC MS/MS) and performed tentative identification of compounds that may contribute to the discrepancy between both methods. For all but one participating laboratory (Data 1) we observed consistent ELISA results lying mostly within 1 SD of the mean 8-oxodG concentration. Mean 8-oxodG levels assessed by ELISA correlated with the data obtained by HPLC MS/MS (R=0.679, p < 0.001). The correlation improved when Data 1 were excluded from the analysis (R=0.749, p < 0.001). We identified three outlying urine samples; one with an ELISA 8-oxodG concentration lower, and two with 8-oxodG levels higher, than those measured by HPLC MS/MS. Omitting these samples further improved inter-methodology agreement (R=0.869, p <0.001). In the outliers with high 8-oxodG estimates various aromatic and heterocyclic compounds were tentatively identified using gas chromatography-mass spectrometry (GC MS). Application of authentic standards revealed the presence of saccharides, including D-glucose and D-galactose as putative interfering substances. In summary, assay standardization improved ELISA inter-laboratory agreement, although some variability is still observed. There are still compounds contributing to overestimation of 8-oxodG by ELISA, but only in some urine samples. Thus, despite significant improvement, ELISA still should not be considered a robust alternative to chromatographic techniques. (C) 2016 Elsevier Inc. All rights reserved.
