AIM: To investigate the effect of 48-hour (h) administration of Tarantula Logoplex (R) (TL), a homeopathic medical product containing Tarantula cubensis venom, alone and in combination with temozolomide (TMZ) on T98G glioblastoma cell line with regard to cytotoxicity, cell migration, nitric oxide synthase (NOS) level, and the type of programmed cell death pathway that mediates this cytotoxic effect. MATERIAL and METHODS: Cytotoxic effect was analyzed using the 3-(4,5-dimethylthiazolyl-2)-2.5-diphenyltetrazolium bromide (MTT) method, apoptosis was analyzed by Annexin V-FITC/PI flow cytometry, autophagic cell imaging was performed using the monodansylcadaverine staining method, mitochondrial membrane potential was evaluated using the tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining method, and cell migration was analyzed using the scratch test. The levels of eNOS, iNOS, and LC3 proteins were evaluated using immunofluorescence (IF) and western blot analyses. Results were compared and statistically evaluated. RESULTS: Annexin V-FITC/PI flow cytometry revealed that the cytotoxicity of the combined administration was high and primarily (37.57%) occurred through apoptosis. According to JC-1 analysis, the apoptotic effect could have originated from mitochondria. Cell migration was lowest at the IC50 dose of TL. The order of fluorescence intensity from the strongest to the weakest was control>TL>combination>TMZ for eNOS and control>TL>combination>TMZ for iNOS. Western blotting revealed the highest eNOS and iNOS protein density with TL IC25 administration and the highest LC3 protein density with TMZ IC50 administration. CONCLUSION: Combined administration of TL and TMZ may exert a significant cytotoxic effect on T98G glioblastoma cells, which may occur through apoptosis. TL may play a role in augmenting the effect of conventional therapeutic drugs on glioblastoma.

AIM: To identify the autophagy mechanism T98 glioma cells. MATERIAL and METHODS: Three groups were created with T98 human glioblastoma cells; Group 1: T98 glioma cells without treatment (Control group). Group 2: T98 glioma cells treated with 3 Nl/ml JWO. Group 3: T98 glioma cells treated with 6 Nl/ml JWO. The cell proliferation, oxidative stress, types of cell death were studied at IC50 dose of JWO. RESULTS: The proliferation of glioma cells was inhibited in 5.296 Nl/ml dose. JWO induced apoptosis in T98 glioma cells in comparison with the control and there was statistically significant difference (p<0.001). Apoptosis was analyzed via TUNEL method and results were checked by flow cytometry. We also investigated the effects of JWO on autophagy in T98 glioma cells by immunostaining LC3-II and MDC fluorescent stainings. The differences between JWO treated and control group were notably significant (p<0.001). The immunofluorescence staining resultsof LC3-II was confirmed by Western blotting analysis. CONCLUSION: JWO seems to be an effective treatment agent for glioblastoma. Not only does it induce apoptosis via oxidative stress but also affects the autophagy. The use of JWO in combination with other treatment options may increase the efficacy of treatment.

In this study, we investigated the effect of doxorubicin and tunicamycin treatment alone or in combination on MDM-, Cul9-and prion protein (PrP)-mediated subcellular regulation of p53 in the context of apoptosis and autophagy. MTT analysis was performed to determine the cytotoxic effect of the agents. Apoptosis was monitorized by ELISA, flow cytometry and JC-1 assay. Monodansylcadaverine assay was performed for autophagy. Western blotting and immunofluorescence were performed to determine p53, MDM2, CUL9 and PrP levels. Doxorubicin increased p53, MDM2 and CUL9 levels in a dose-dependent manner. Expression of p53 and MDM2 was higher at the 0.25 μM concentration of tunicamycin compared to the control, but it decreased at 0.5 μM and 1 μM concentrations. CUL9 expression was significantly decreased only after treatment of tunicamycin at 0.25 μM. According to its glycosylation status, the upper band of PrP increased only in combination treatment. In combination treatment, p53 expression was higher than control, whereas MDM2 and CUL9 expressions were decreased. Combination treatments may make MCF-7 cells more susceptible to apoptosis rather than autophagy. In conclusion, PrP may be important in determining the fate of cell death through crosstalk between proteins such as p53 and MDM2 under endoplasmic reticulum (ER) stress conditions. Further studies are needed to obtain in-depth information on these potential molecular networks.

Programlanmış tip II hücre ölüm tipi olan otofaji kendi kendini yeme işlemidir. Açlık ve diğer stres durumlarında biyomoleküllerin yapı taşlarının bazal seviyelerini koruyan hücre içi bir geri dönüşüm sürecidir. Lizozom aracılı katabolik bir süreç olan otofaji hücresel homeostazın sürdürülmesinde önemli bir role sahiptir. Otofaji tiplerinden biri olan makrootofaji yolağı, iç ve dış sinyallerin uyarımı sonucunda hücre içi sindirilecek materyal (yanlış katlanmış ya da bozulmuş proteinler, hasarlı organeller, vb.) otofagozom içine alınarak, otofagozomun lizozomla birleşmesi sonucu lizozomal aktivite ile sindirilmesi sürecini içerir. Otofaji sinyal yolağı otofaji ile ilişkili genler (ATG’ler)’in ürünleri ile ilave proteinler ve otofaji ile ilişkili kinazlar tarafından düzenlenmektedir. Otofaji mekanizmasındaki bozukluklar kanserin de dahil olduğu çeşitli hastalıklarla ilişkilendirilmektedir. Tümör oluşumu sırasında otofajinin kanserin erken evresinde tümör baskılayıcı, ileri evrede ise tümörü teşvik edici ikili bir role sahip olduğu çeşitli çalışmalarla ortaya konmuştur. Otofajinin karmaşık yapısı ve kanserdeki bu ikili rolü sebebiyle mekanizmanın tam olarak aydınlatılması ve farklı kanserlerde hangi aşamada nasıl bir role sahip olduğunun belirlenmesi ya otofaji baskılayıcı ya da otofajiyi teşvik edici etkin tedavilerin geliştirilmesine olanak tanıyacaktır. Bu derlemede, makrootofaji sinyal yolağının moleküler mekanizması, otofajinin kanserdeki ikili rolü ve otofaji ile ilişkili kansere karşı geliştirilen tedavi yaklaşımları hakkında bilgiler verilmesi amaçlanmıştır.

In this study, the purpose was to investigate the histopathological, genotoxic effect, oxidative stress and cell death due to Metronidazole (MTZ), which is a 5-nitroimidazole compound, used widely for the treatment of anaerobic organism infections in fish and humans on gill and liver tissues of Oncorhynchus mykiss. Trout fishes were exposed to 5, 10, and 20 mg/L of MTZ in the aquariums for 2, 4 and 8 days. Staining technics namely H&E, NOS immunohistochemistry, and TUNEL were performed to determine histopathological changes, oxidative damage and apoptosis. Additionally, smear preparations were also prepared from gill blood for genotoxic evaluations. The organ damage started in the 2(nd) day with 5 mg/L MTZ application and effects increased per duration and dose-dependent manner. It was observed that the gills had the primary and secondary lamellae lengths, with formation of clavate lamellae, fusion in secondary lamellae, separation of epithelium and aneurysm. Regional necrosis, vacuolization of hepatocytes, pycnotic nucleus, enlarged sinusoids were also determined in the liver. NOS immunoreactivity increased with the inducible immunoreactivity (iNOS) that was more prominent when compared to the endothelial immunoreactivity (eNOS). Apoptotic immunoreactivity was higher in the 10 mg 8(th) day experimental group at liver and gills, and was lower 20 mg 8(th) day experimental group. When the gills and liver compared with each other, in all doses, immunoreactivity was lower in gills, compared with liver. Genotoxic examinations showed that both number of micro nucleated erythrocytes and nuclei abnormalities were higher in MTZ-treated groups.

We identified the developmental stages of the chicken lung, and explored the histopathological effects of L-nitro-arginine-methyl-ester (L-NAME) on such development at different times. L-NAME was injected into the vitellus, at two different doses, on the day of embryogenesis 6, 8, 9, 11, 13, and 16. Sham-injected and experimental embryos were collected on the day of incubation 7, 9, 10, 12, 14, and 17. Chicken lungs developed in four stages: embryonic (4–7 days), pseudoglandular (8–14 days), canalicular (14–17 days), and saccular (or alveolar) (day 17 and postnatally). Most differentiation occurred in the pseudoglandular stage. L-NAME triggered mesenchymal tissue loss, reduced airway branching and lung volume, narrowed bronchial diameters, triggered formation of pulmonary emboli, enhanced alveolar protein accumulation, caused regional bleeding (hemorrhage), triggered abnormal blood vessel modeling, and reduced vessel diameter. Sham-injected embryos exhibited strong immunoreactivities against endothelial and inducible nitrous oxide synthase in the embryonic and canalicular stages; immunoreactivities were reduced at all developmental stages in the experimental group.

Objective: This study examined the development of nephrons and the differentiation of juxtaglomerular apparatus (JGA) cells in BALB/c type mouse embryos. Thick and thin epon sections were investigated by light microscopy (LM) and transmission electron microscopy (TEM) respectively. Materials and Methods: Nephron development is completed by passing through the nephrogenic vesicles in the respected stages of comma shape body, S-shape body, precapillary, immature glomerular, and mature glomerular stage. JGA is distinguished in the mature glomerular stage of nephron development at LM level. According to TEM observations, signs of differentiation of juxtaglomerular (JG) cells and macula densa (MD) cells forming JGA are seen earlier, in precapillary stage. In developed Golgi fields that characterize JG cells, large number of electron lucent small vesicles and small number of electron dense large renin granules are seen in the precapillary stage. In the mature glomerulus, the number of electron dense large granules increases in JG cells. The differentiation of MD cells is indicated by the formation of primary cilia on their apical faces in precapillary stage. Deep membrane folds are formed in the basal and lateral faces of MD cells in the mature glomerular stage. There are many numbers of large mitochondria in these regions. Results: JG and MD cells, which play an absolute role in the regulation of the renin-angiotensin system are differentiated in precapillary stage before completion of nephron development and formation of blood vessels. Conclusion: Such early differentiation of JGA cells suggests that the renin-angiotensin system is important both in the development of the kidney and in the total development of embryo.

AIM:To evaluate the neuroprotective effects of deocanthal OC in a rat model of traumatic brain injury (TBI).MATERIAL AND METHODS:Twenty-six adult male, Wistar albino rats were used. The rats were divided into 4 groups. Group 1 was the sham group (n=5). Group 2 was the trauma group (n=5) where rats were treated with 10 mg/kg saline intraperitoneally (IP) twice a day. Groups 3 and 4, rats were treated with 10 (group 3, n=8) or 30 (group 4, n=8) mg/kg OC IP twice a day. For each group, brain samples were collected 72 hours after injury. Brain samples and blood were evaluated with histopathological and biochemical methods.RESULTS:Histopathological evaluation revealed a significant difference between Group 2 and Group 4. Biochemical findings demonstrated that the oxidative stress index was highest in Group 2 and lowest in Group 4.CONCLUSION:OC has a protective effect on neural cells after TBI. This effect is achieved by reducing oxidative stress and apoptosis.

Antiepileptic drugs (AED) are teratogens that confer a risk of various congenital malformations including neural tube defects. Lacosamide (LCM) is a novel third-generation AED and its effects on neural tube (NT) development remain unclear. Thus, the aim of the present study was to investigate the effects of LCM in the early stages of NT development in the chicken embryo. Three different doses of LCM were applied under the embryonic disks of chicken embryos after they were incubated for 30 hours. Incubation was continued for an additional 80 hours and then all embryos were obtained for routine histology. In the LCM-treated groups, there was occlusion in the middle and ventral levels of the cavity. In the group treated with 1.60 mg of LCM, the NT cavity was closed, the notochord exhibited deterioration, and cellular association appeared abnormal. In the groups treated with 0.12 and 0.5 mg of LCM, the ectoderm layer surrounding the embryo was hypertrophic and the number of pyknotic cells increased in a dose-dependent manner. Thus, different doses of LCM had different effects. The results of this study demonstrated that LCM causes histopathological alterations during the neurulation stage that might ultimately result in the development of congenital defects and/or malformations.

Metronidazole (MTZ) is commonly used as an antibiotic for enterozoa; however, its side-effects have not been fully characterized. In this study, the toxic effect of a dose and time-dependent MTZ application on morphology of the intestinal tissue of Oncorhynchus inykiss was examined. Fish were exposed to 5, 10, and 20 mg/L of MTZ in the aquariums for 2, 4 and 8 days. Samples taken from the jejunum region of the small intestine were for Gomori Trichrome, NOS and TUNEL. A slight toxic effect that started with a dosage of 5 mg/L for 2 days reached the maximum level with a dosage of 20 mg/L for 8 days. Erosion, oedema, inflammation, disintegration in the epithelium, and necrosis were detected at the tips of the villi. NOS staining had increased a great deal. Separately, a parallel TUNEL staining was monitored in the process of NOS staining practices. The toxic effect that increased in proportion to the dosage and period had occurred due to oxidative damage and that the cells had been induced to die through apoptosis. It was thought that this toxic effect could be of significance because of food chain and needed to be investigated by means of advanced techniques.

Objective: Alcohol continues to be consumed even though its harmful effecs are well established. One of the most common damage of alcohol consumption is fetal alcohol syndrome, characterized by craniofacial anomalies, cardiac anomalies and neural tube defects. Therefore, understanding the molecular mechanisms underlying the alcohol-induced toxicity that occur with time and dose dependent manner is very important., Most of the studies in order to understand the effects of alcohol have been carried out on early neurulation, however its effects on late neurulation are still unknown. Therefore in this study, effects of alcohol on secondary neurulation were investigated in chick embryos. Methods: Leghorn breed of embryonic chicken eggs were used. At 50 h of incubation, 100 mu L 50% ethanol solution was injected. Depending on the period of exposure to alcohol, varying degrees of pathological disorders were detected in E3, E7 and E10 days. Results: Developmental delay, structural abnormalities, morphological abnormalities in the heart and face and especially presence of two spinal cord cavities were found. In addition, we also detected delays in the closure of the neural tube, cellular deformities and the structural abnormalities in notochord. While eNOS, iNOS, and TUNEL levels increased, while laminin levels decreased. Conclusion: In this study during late development, significant alcohol-induced morphological and histopathological changes were observed. We also determined Increased level of oxidative stress caused by alcohol was accompanied with the changes in matrix composition. Better understanding of these mechanisms which affect the cell behavior is important and will allow learning of harmful effects of alcohol.

At the embryonic development, signal transduction pathways, genetic factors, involvements between nucleus and cytoplasm, environmental factors, cell-cell and cell-matrix interactions have important roles. It has been known that the cells are regulating the Extracellular Matrix (ECM) synthesis, degradation and reshaping events, also it has been known that the Nitric Oxide is an important molecule for cellular communication and have effects on ECM molecule distribution by reacting with ECM molecules. In this reason, the purpose of our study is detecting the correlation of reactive nitrogen species with a glycosylated molecule laminin α1. In this experiment, Leghorn type SPF (Specific Pathogen Free) embryonic chick eggs has been used. Embryos are collected at 5 th , 6 th and 7 th days at incubation and taken into the 10% neutral buffered formalin solution. The liver tissues that dissected from embryos are fixated at second time. After 24 hours, it has been subjected to the routine paraffin embedding method and embedded to paraffin. From the 5µm sections, immunohistochemistry for eNOS, iNOS and Laminin α1 distribution, and for general histologic evaluation, Haematoxylene-Eosin stains has been applied. eNOS and iNOS immunoreactivity has been observed at peripheral zone of developing liver tissue that epithelial-mesenchymal transition takes place. It has been determined that immunoreactivity was minimal in 5 th day, increasing with the days progressed and have highest at 7 th day. Also, eNOS staining has been more powerful than iNOS staining. Laminin immunoreactivity has been similar at all developmental stages, but relatively, has been showed lesser staining. Particularly, the presence at the zones of cell differentiation has been noteworthy.

Metranidazole (MTZ) is an antibiotic used for parasitic infections in a number of species. Accumulation of this drug in the environment and its interaction with fish of economic value makes this drug particularly important. In the present study, we examined the histopathological effects of MTZ on the intestinal tissue of Oncorhynchus mykiss. The fish in aquarium were exposed to MTZ at doses of 5, 10, 20 mg/L for 2, 4 and 8 days. At the end of the experiments, macroscopic pathology or death were not observed at these doses. Histochemical staining with Haematoxylene-Eosin, Periodic Acid Schiff and Gomori Trichrome showed, depending on increased dose and prolonged duration, areas of necrosis, edema, inflammation, small tears at the tips of the villi and excretion with heterogenic distribution of the Goblet cells. Moreover, changes in the connective tissue of the intestines due to toxicity of MTZ and decreases in immunostaining of matrix proteins such as laminin and collagen IV, especially in the epithelium were observed. Findings of the present study would be useful to demonstrate the adverse effects of MTZ use, emphasizing the importance of the effect on fish which could be very important public health.

Antiepileptic drugs (AEDs) are teratogens and confer a risk of congenital malformation. The estimated prevalence of major congenital malformations such as cardiac defects, facial clefts, hypospadias, and neural tube defects in epileptic women is 4–10 %, which represents a two- to fourfold increase in pregnant women compared to the general population. However, there are no clear data for newer drugs. Lacosamide (LCM), a novel AED, is the first of the third-generation AEDs to be approved as adjunctive therapy for the treatment of partial-onset seizures. There are no data on the pharmacokinetics of LCM during pregnancy, and only some published data have reported on its effects during pregnancy.

In this study, we investigated the distribution of laminin, collagen type IV, nidogen and fibronectine during metanephric development in fetal mouse kidney by immunohistochemistry. Stain density of basement membranes of tubules, glomerules and mesangial matrix were compared in pre-capillary, immature glomerular and mature glomerular stages of fetal kidney.All the matrix proteins were strongly stained in precapillary stage.In immature glomerular stage, a strong staining was observed for fibronectin.Staining intensity was slightly decreased for the other proteins in this stage.In mature glomerular stage, diminished staining for all proteins was observed similar to the previous stage, except fibronectin.The strongest immunoreactions were found for fibronectin and nidogen in all investigated stages.In general, there was a similar staining intensity for all glycoproteins during maturation except for laminin.It was thought that the distribution of extracellular matrix molecules plays an important role for the kidney development.Interactions amoung these molecules probably crucial on cell behavior like migration, proliferation and differentiation in normal development of the nephron.

The microscopic structures of the liver tissues from two amphibian species, T. vulgaris and T. karelinii , were studied by different histological staining techniques. The polyhedral shaped parenchymal cells of T. karelinii contain euchromatic mononuclei and dense cytoplasmic glycogen. These cells arranged as systematical cell cords in parenchyma. Pigment granules containing Kupffer cells (melanomacrophages) were found in the sinusoid wall. T. karelinii liver parenchyma have shown no clear lobulation and no concentrated reticular fiber network around the hepatocytes and vessels. The predominant glycosaminoglycan (GAG) type of T. karelinii was hyaluronic acid (HA), however, sulphated GAGs were almost absent. Hepatocytes from T. vulgaris were not polyhedral shaped, and contain euchromatic mononucleus. The glycogen content was not significant quantities in hepatocyte cytoplasm of T. vulgaris . These cells form radial arrangenment close to large vessels, however they found as cell clusters in bottom of parenchyma. Kupffer cells were found between the parenchymal cell groups and at the sinusoidal walls. These cells contain many pigment ganules in their cytoplasms. Reticular fibre networks were found between hepatocytes and vessels in lobulation lacking parenchyma. Around the hepatocytes and vessels, hyaluronic acid was the predominant GAG type.

Histological structures of esophagus and stomach tissue samples of Lacerta stellio have been studied, and glycosaminoglycan (GAG) distribution has been histochemically determined. Histologically, esophagus and stomach of L. stellio are composed of four layers: mucosa, submucosa, muscularis mucosae and serosa. Mucosa of esophagus is covered by simple columnar ciliated epithelium with many mucous secreting goblet cells and contains branched tubular glands. Stomach of L. stellio is composed of fundus (oral and aboral) and pylorus regions. Mucosa is covered by columnar epithelium. Fundic glands are branched tubular glands while pyloric glands are usually simple tubular glands. In both regions of the stomach, glands are subdivided into three areas as base, neck and isthmus. Both in the esophagus and stomach, muscular layer is in the form of smooth muscle having inner circular and outer longitudinal layers. According to the results obtained by Alcian Blue (pH 5.8)/Periodic Acid Schiff staining, stomach is similar to esophagus in that neutral mucins and hyaluronic acid (HA) are dominant in isthmus and neck regions of gland tissue of stomach. In the base of the stomach, only neutral mucins have been observed. HA has been observed to be dominant in all other regions of both stomach and esophagus, along with some but not much sulphated GAGs.

Alcohol as a teratogenic agent inhibits cell growth, function, proliferation and migration by affecting macromolecules, and can induce cell death. Prenatal ethanol exposure causes neural tube defects (NTD) and growth deficiency in experimental animals. NTDs are a group of malformations that result in failure of neural tube (NT) closure in early embryonic development and are among the most common congenital malformations in humans. NTDs are also associated with a number of other central nervous system malformations. Basal layers are the most densely stained structures with Alcian blue which determines glycosaminoglycan (GAG) types. While all sulphated GAGs were observed in the basal layers of NT of the embryos in control and saline-injected groups, hyaluronic acid was dominant in the 10% alcohol-administered embryos. It was reduced in the 15% alcohol-administered embryos and keratan sulphate was significantly low in 20% samples. Especially in the control and saline-injected groups, chondroitin sulphate and dermatan sulphate were highly expressed around cells migrating from the NT, while the same were reduced in 10% alcohol-administered embryos. In 15% alcohol-administered embryos, while the heparine and heparane sulphate were dense around cells migrating from the NT, staining specificities were decreased in 20% alcohol-administered embryos in same regions. Increased alcohol degrees cause decrease of the GAG types in both areas.

The aim of the present study was to determine heavy metal accumulation in water, sediment and some tissues of Anguilla anguilla along with determining histopathological and genotoxic effects of accumulation on these tissues by using light microscopy. Water, sediment and fish tissue samples taken from different sites of 3 different study areas were studied and the order of accumulation of metals was Fe>Pb>Mn>Co>Zn>Ni> Cr>Cu>Cd in water, Fe>Co>Mn>lpb>Zn>Ni>Cr>Cu>Cd in sediment and Cd>Mn>Cu>Cr>Ni>Fe>Zn>Co>lpb, Cd>lpb>Ni>Cr>Mn>Cu>Fe>Co>Zn and Cr>Ni>Zn>Cd>lpb>Mn>Cu>Fe>Co in liver, muscle and gill of fish, respectively. In histopathological studies, a decrease in the length of primary and secondary lamellae of gills, fusion in secondary lamellae, cellular proliferation, clavate lamellae formation and necrosis were observed. In liver tissue, dilation of sinusoid, increase in the number of erythrocytes, ruptured hepatocytes, decrease in glycogen accumulation and vacuolization were observed. In muscle tissue, necrosis, cellular dissolution and loss of striatation in muscle fibers were found. It was observed that pollution of water had no genotoxic effect on Anguilla anguilla.