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Background: Among the many types of chemical scaffolds, isatin derivatives, including their Schiff bases, have been extensively studied to find novel therapeutic agents against cancer. Amide or urea groups containing derivatives were also discovered to be tyrosine kinase inhibitors. Objective: This study aims to find potent compounds by designing 16 novel urea and sulfonamide derivatives of isatin Schiff bases. Methods: Compounds were tested against PC-3, HepG2, SH-SY5Y, A549 cancerous, and NIH/3T3 non-cancerous cell lines using cell culture assay. Results: Among the tested compounds 7a, 7b, 7c, 7d, 7h, 8a, and 8f presented potential inhibitions against cellular proliferation activities of HepG2 cells with average IC50 values of 31.97, 42.13, 31.50, 47.98, 32.59, 43.44, and 37.81 mu M, respectively. They showed better inhibition potencies than the reference compound doxorubicin, and its value was measured as 51.15 mu M in the same culture assay. The cytotoxic activities of the compounds in other cell lines were found to be less potent compared to doxorubicin. Conclusion: In vitro experiments demonstrated that designed compounds have the first evidence that they might be active against hepatocellular carcinoma. According to ADME prediction results, all compounds presented drug-like and good metabolic properties.

Benomyl, benzimidazole group pesticide, has been prohibited in Europe and USA since 2003 due to its toxic effects and it has been still determined as food and environmental contaminant. In the present study, the toxic effect mechanisms of benomyl were evaluated in rat cardiomyoblast (H9c2) cells. Cytotoxicity was determined by MTT and NRU assay and, oxidative stress potential was evaluated by reactive oxygen species (ROS) production and glutathione levels. DNA damage was assessed by alkaline comet assay. Relative expressions of apoptosis related genes were evaluated; furthermore, NF-kappa B and JNK protein levels were determined. At 4 mu M concentration (at which cell viability was >70%), benomyl increased 2-fold of ROS production level and 2-fold of apoptosis as well as DNA damage. Benomyl down-regulated miR21, TNF-alpha and Akt1 > 48.75 and > 97.90; respectively. PTEN, JNK and NF-KB expressions were upregulated. The dramatic changes in JNK and NF-kappa B expression levels were not observed in protein levels. These findings showed the oxidative stress related DNA damage and apoptosis in cardiomyoblast cells exposed to benomyl. However, further mechanistic and in vivo studies are needed to understand the cardiotoxic effects of benomyl and benzimidazol fungucides.

Zearalenone, produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin that contaminates cereals, resulting in adverse effects on human health. We investigated the effects of zearalenone and its metabolite alpha zearalenol on epigenetic modifications and its relationship with metabolic pathways in human hepatocellular carcinoma cells following 24 h of exposure. Zearalenone and alpha zearalenol at the concentrations of 1, 10 and 50 mu M significantly increased global levels of DNA methylation and global histone modifications (H3K27me3, H3K9me3, H3K9ac). Expression levels of the chromatin modifying enzymes EHMT2, ESCO1, HAT1, KAT2B, PRMT6 and SETD8 were upregulated by 50 mu M of zearalenone exposure using PCR arrays, consistent with the results of global histone modifications. Zearalenone and alpha zearalenol also changed expression levels of the AhR, LXR alpha, PPAR alpha, PPAR gamma, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPAR gamma, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPAR gamma promoter reduced methylation significantly following zearalenone exposure. Taken together, the epigenetic mechanisms of DNA methylation and histone modifications may be key mechanisms in zearalenone toxicity. Furthermore, effects of zearalenone in metabolic pathways could be mediated by epigenetic modifications.

Zearalenon (ZEA), çeşitli Fusarium türleri tarafından üretilen steroid olmayan östrojenik bir mikotoksindir. Mısır, buğday, yulaf ve soya fasülyesi gibi tahıl ürünlerini ve suları kontamine ederek insan sağlığı üzerine olumsuz etkilerde bulunur. Doktora tez çalışmasında bir endokrin bozucu olan ZEA ve metaboliti α-ZOL'ün epigenetik modifikasyonlar ve metabolik yolaklar üzerine etkisi HepG2 hücrelerinde incelenerek metabolik fonksiyon bozukluklarıyla muhtemel ilişkisi aydınlatılmaya çalışılmıştır. HepG2 hücrelerinde MTT ve NRU testlerine göre IC50 değerleri sırasıyla ZEA için 143,35 ve 60,45 µM; α-ZOL için ise 111,42 ve 35,73 µM olarak belirlendi. BrdU testi ile hücre proliferasyonunda 1-50 µM konsantrasyonlarda 24 saat ZEA maruziyeti sonrasında %7,2-41,71 oranında; 24 saat α-ZOL maruziyeti sonrasında ise %11,78-44,33 oranında kontrole kıyasla anlamlı artış tespit edildi. ROS oluşumunda 50 µM ZEA ve 50 µM α-ZOL maruziyeti sonucunda %48,1 ve %49,6 oranında kontrole kıyasla anlamlı artış gözlendi. Global DNA metilasyonu seviyelerinde 10 ve 50 µM ZEA maruziyeti 1,46 ve 2,54 kat; 1-50 µM konsantrasyonlarda α-ZOL maruziyeti ise 1,53-1,58 kat anlamlı artışa neden oldu. 1-50 µM konsantrasyonlarda ZEA ve α-ZOL maruziyeti PPARɣ ekspresyonu ve promotör DNA metilasyon seviyelerinde anlamlı düzeylerde değişikliklere neden olmuştur. Aynı zamanda, 1-50 µM konsantrasyonlarda ZEA ve α-ZOL maruziyetinin nükleer reseptör genleri (AhR, LXRα, PPARα, PPARγ), karbonhidrat ve lipit metabolizmasında rol oynayan genler (L-fabp, LDLR, GAPDH, Glut2, Igf-1, Akt1, HK2) ve oksidatif hasar ile ilişkili genlerin (HO1, Hsp70, Nrf2) ekspresyon seviyelerini önemli derecede değiştirdiği gösterildi. Ayrıca, 1-50 µM konsantrasyonlarda ZEA ve α-ZOL maruziyetinin western blot tekniği ile global histon modifikasyonları (H3K27me, H3K9me3, H3K9ac) üzerinde anlamlı değişikliklere neden olduğu görüldü. Tez çalışmasından elde edilen sonuçların, endokrin bozucu kimyasalların toksisitesinde ortaya çıkan anahtar moleküler mekanizmaların daha iyi anlaşılmasına katkı sağlaması beklenmektedir.Anahtar Kelimeler: Zearalenon; α-zearalenol; epigenetik mekanizmalar; nükleerreseptör genleri; karbonhidrat ve lipit metabolizması; oksidatif hasar. Zearalenone (ZEA), produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin. It contaminates cereals such as corn, wheat, oat and soybean and water, therefore it has negative effects on human health. In doctorate thesis, the effects of ZEA and its metabolite α-ZOL on epigenetic modifications and metabolic pathways have been investigated in HepG2 cells in order to elucidate the possible relationship between metabolic dysfunctions. According to the MTT and NRU tests, the IC50 values were determined as 143.35 and 60.45 for ZEA and 111.42 and 35.73 µM for α-ZOL, respectively. At the concentrations of 1-50 µM of ZEA and α-ZOL exposures for 24 h were significantly increased cell proliferation by BrdU assay in the rates of 7.2-41.71% for ZEA, 11.78-44.33% for α-ZOL, respectively, compared to the control. 50 µM of ZEA and α-ZOL exposure significantly increased levels of ROS generation in the rates of 48.1% and 49.6%, respectively, compared to the control. ZEA caused an increase in global DNA methylation levels at 1.46 and 2.54 fold after 10 and 50 µM of ZEA exposure and at 1.53-1.58 fold after 1-50 µM α-ZOL exposure. 1-50 µM of ZEA and α-ZOL exposure caused alterations in PPARɣ expressiom and promoter DNA methylation significantly. Besides, 1-50 µM of ZEA and α-ZOL exposure showed significant alterations in the expression levels of nuclear receptor genes (AhR, LXRα, PPARα, PPARγ), carbohydrate-lipid metabolism related genes (L-fabp, LDLR, GAPDH, Glut2, Igf-1, Akt1, HK2), and oxidative stress genes (HO1, Hsp70, Nrf2). In addition, 1-50 µM of ZEA and α-ZOL exposure resulted in significant changes in global histone modifications (H3K27me, H3K9me3, H3K9ac) by western blot technique. It is expected that results from this thesis will contribute to better understanding of key molecular events in the toxicity of endocrine disrupting chemicals for risk assessment process.Key Words: Zearalenone; α-zearalenol; epigenetic mechanisms; nuclear receptor genes;carbohydrate and lipid metabolism; oxidative damage. 191

Bisphenol A (BPA), as synthetic monomer used in industry in theproduction of polycarbonate plastic and epoxy resins, has endocrine disruptorproperties and high risk on human health. Continuous release of free BPA intofood, beverages, and the environment has resulted in a widespread human exposureto this chemical. Recent studies have showed the role of endocrine effects ofenvironmental chemicals on the changes in gene expression may be associatedwith epigenetic mechanisms such as DNA methylation and histone modifications.The aim of the study was to investigate dose-related effects of BPA (0, 0.1 μM,1 and 10 mM for 48 and 96 h) on global and gene-spesific (p16, cyclin D2 andRassf1 genes) DNA methylation in human hepatocarcinoma (HepG2) cells.We also investigated global histone modifications such as H3K9 trimethylation(H3K9me3), H3K9 acetylation (H3K9ac), H3K27 trimethylation (H3K27me3).The 50% inhibitory concentration (IC50) value of BPA was determined as 134 and180 μM in HepG2 cells for 24 h by MTT and neutral red uptake (NRU) tests,respectively. We observed decrease on the global levels of 5-methylcytosine and5-hydroxymethylcytosine at 1 and 10 mM after 96 h BPA exposure. There is nosignificant alterations on the promoter-region of methylation and expression ofp16, cyclin D2 and Rassf1 genes. Global levels of H3K9me3 decreased after 0.1and 1 μM concentration of BPA exposure for 48 h, while increased after 96 h 0.1and 1 μM of BPA exposure for. Hovewer, it has not been observed significantlychanges for the global levels of H3K27me3 and H3K9ac. In this study we suggestthat BPA may disrupt epigenetic events by altering global and gene-spesific DNAmethylation and histone modifications in HepG2 cells.

Enoksolon (18-ß Glisirretinik asit), meyan bitkisinden (Glycyrrhiza glabra L.) elde edilen pentasiklik triterpenlerden biridir. Meyan yüzyıllardır terapötik özellikleri sayesinde özellikle Asya?da geleneksel tıpta kullanılmıştır. Enoksolonun pek çok farmakolojik özelliği vardır. Antiinflamatuvar, antitumoral, antihepatotoksik aktivite gösterir. Kozmetiklerde akne preparatlarında, güneş sonrası losyonlarında, leke giderici ürünlerde ve selülit kremlerinde diğer maddeler ile kombine halde piyasaya sürülmektedir. Bu çalışmada, yaraların iyileştirilmesinde kullanılmak üzere farklı taşıyıcılar içinde enoksolonun topikal preparatları (jel, yağlı krem ve Y/S emülsiyon) hazırlanmıştır. Bu amaçla, öncelikle enoksolonun fizikokimyasal özellikleri tayin edilerek, miktar tayini yöntemi belirlenmiş ve çözünme ortamındaki çözünürlüğü tayin edilmiştir. Hazırlanan topikal preparatlar 12 ay süresince oda sıcaklığı, 4oC ve 40oC?de karanlıkta bekletilerek fiziksel kararlılık çalışmasına tabi tutulmuştur. Yapılan in vitro salım çalışmaları ile formülasyonların enoksolonu salım özellikleri araştırılmıştır. Sonuç olarak, penetrasyon arttırıcılardan oleik asit (OA), limonen (L) ve izopropil miristatın (IPM) ksantan jeli ile geçimsiz olduğu ve beraber kullanılamayacağı sonucuna varılmıştır. Aynı durumun yağlı krem için, L ve IPM ile söz konusu olduğu görülmüştür. PG?ün ise jel formülasyonunun salım hızını azalttığı dikkat çekmiştir. PG ve OA?in ise, yağlı kremin enoksolonun salım hızını arttırdığı tespit edilmiştir. IPM?ın Y/S emülsiyonu içindeki konsantrasyonu arttıkça salım özelliklerini düzelttiği, PG, OA ve L?in yine salım hızında artışa neden olduğu görülmüştür. Enoxolone (18-ß Glycyrrhetinic acid) is one of the pentacyclic triterpenes which is obtained from licorice (Glycyrrhiza glabra L.). Licorice has been used for traditional medicine particularly in Asia for centuries. Enoxolone has a lot of pharmacological properties. It displays antiinflammatory, antitumoral and antihepatotoxic activities. It is available in acne preparations, after-sun lotions, skin whitenning products and cellulite creams in the commerce.In this study, topical preparations of enoxolone in different vehicles (gel, oleageneous cream and O/W emulsion) were formulated to be used for wound healing. For this purpose, physico-chemical properties of enoxolone were determined, its analytical quantification method was defined and its solubility in the release medium was assayed. Physical stability study was employed storing topical preparations at the room temperature, 4oC ve 40oC in the dark for 12 months. Release properties of formulations were investigated with in vitro release studies. As a result, oleic acid (OA), limonene (L) and isopropyl myristate (IPM) as penetration enhancers were found incompatible with the gel of xanthan gum and that they could not be used together. It was seen that the same situation is available with L and PM in the case of oleageneous cream. PG was seen that it increased release rate of gel formulation. In the case of oleageneous cream, PG and OA were detected to increase release rate of enoxolone. It was found that increase in the rate of IPM in O/W emulsion was improved release properties of the formulation when PG, OA and L caused increase in the release rate. 112