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Öğe Alfa amanitinin sıçanlarda toksikokinetiği(Düzce Üniversitesi, 2012) Karahan, Selim; Kaya, ErtuğrulALFA AMANİTİNİN SIÇANLARDA TOKSİKOKİNETİĞİAmanita phalloides dünyadaki görülen ölümcül mantar zehirlenmelerinin %95'in üzerinde sorumlu olan mantar türüdür. Bu mantarın 2 grup toksini tanımlanmıştır. Bunlar amanitin grubu ve falloidin grubu toksinler olarak bilinmektedir. Amanitin grubu toksinlerden mantar içinde en fazla bulunan ve hakkında en fazla araştırma yapılan toksin ?-amanitindir. Bu grup toksinler RNA polimeraz II enziminin spesifik inhibitörüdür. Mantarlarla olan zehirlenmelerde bu toksinin toksikokinetiği çok önemlidir. Ancak bu konuda yapılan araştırmalar yetersizdir.Yaptığımız saha çalışmalarında Ekim-Kasım 2010 aylarında Düzce çevresindeki ormanlardan yeterli miktarda Amanita phalloides mantarı toplanmıştır. Mantar içeriğindeki toksin analizi ve mantarlardan ?-amanitin saflaştırılması HPLC cihazıyla gerçekleştirilmiştir. Çalışmamızda, anestezi altındaki sıçanlara oral ve intravenöz yoldan 1 mg/kg ?-amanitin verildikten sonra farklı zaman dilimlerinde (2.-5.-10.-15.-20.-45.-60.-90. dakika ve 2.-3.-4.5.-6.-9.-12.-24 saat) alınan kanların serumlarında ?-amanitin toksininin HPLC yöntemiyle C18 kolon, amonyum asetat: asetonitril: metanol (80: 10: 10,v/v/v) mobil faz ve 1 mL/dak izokratik akış hızı kullanılarak analizi yapıldı.Yapmış olduğumuz analizler sonucunda, ?-amanitin toksikokinetiğinin ?iki kompartmanlı dışarıya açık model?e uygunluk gösterdiği, oral biyoyararlanımının F=0.936, tdoruk: 2-3 saat (2.64), Cdoruk-oral = 2.86 ± 0.08 µg/ml, Cdoruk-i.v. = 12.01 ± 0.07 µg/ml, klirensinin CL = 75.91 ± 9.11 ml/st/kg ve eliminasyon yarılanma ömrü (t1/2ß) 110.4 ± 0.30 dak. olduğu saptandı.Çalışmamızda oral biyoyararlanım oranı, eliminasyon yarılanma ömrü, tdoruk, Cdoruk, klirensin belirlenmesi ve literatüre kazandırılması önemli bir aşamadır. Ayrıca bu verilerden zehirlenme vakalarında yararlanılabilecektir.Anahtar sözcükler: Amanita phalloides, ?-amanitin, HPLC, toksikokinetik, biyoyararlanım, klirensÖğe Amanitin and phallotoxin concentration in Amanita phalloides var. alba mushroom(Pergamon-Elsevier Science Ltd, 2013) Kaya, Ertuğrul; Yılmaz, İsmail; Sinirlioğlu, Zeynep Aydın; Karahan, Selim; Bayram, Recep; Yaykaşlı, Kürşat Oğuz; Severoğlu, ZekiAlthough rarely seen, Amanita phalloides var. alba, a variety of A. phalloides type mushrooms, causes mushroom poisoning resulting in death. Since it is frequently confused with some edible mushrooms due to its white colored cap and macroscopic appearance, it becomes important in toxicological terms. Knowledge of the toxin amount contained in this mushroom type is invaluable in the treatment of cases involving poisoning. In this study, we examined the toxin levels of various parts of the A. phalloides var. alba mushroom growing Duzce region of Turkey. Toxin analyses were carried out for A. phalloides var. alba, which were collected from the forests Duzce region of Turkey in 2011, as a whole and also separately in its spore, pileus, gills, stipe and volva parts. The alpha amanitin, beta amanitin, gamma amanitin, phalloidin and phallacidine analyses of the mushrooms were carried out using the RP-HPLC method. A genetic analysis of the mushroom showed that it had similar genetic characteristics as A. phalloides and was a variety of it. The lowest toxins quantity was detected in spores, volva and stipe among all parts of the mushroom. The maximum amount of amatoxins was measured in the gills. The pileus also contained a high amount of amatoxins. Generally, amatoxins and phallotoxin concentrations were lower as compared to A. phalloides, but interestingly all toxins other than gamma toxin were higher in the spores of A. phalloides var. alba. The amount of toxin in all of its parts had sufficient concentrations to cause death. With this study, the amatoxin and phallotoxin concentrations in A. phalloides var. alba mushroom and in its parts have been revealed in detail for the first time. (C) 2013 Elsevier Ltd. All rights reserved.Öğe Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues(Sage Publications Inc, 2015) Kaya, Ertuğrul; Karahan, Selim; Bayram, Recep; Yaykaşlı, Kürşat Oğuz; Çolakoğlu, Serdar; Sarıtaş, AyhanMost of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure -, -, and -amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Duzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. -, -, and -Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 x 4.6 mm; 5 mu m particle) was used. The least amount of -amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.Öğe Caffeine Levels in Turkish Coffee and Some Beverages Sold in Turkey(Düzce Üniversitesi, 2013) Hancı, Mustafa; Bayram, Sait; Karahan, Selim; Kaya, Ertuğrul; Bakırcı, SinanAim: Caffeine has a stimulating effect on the central nervous system functions and are includednaturally in beverages such as tea and coffee while it is added later some beverages such ascola. Different amounts of caffeine are consumed with these drinks in many countries. Inparticular, the amount of caffeine in Turkish coffee is not clear. In our study, caffeine levelswere measured at beverages and Turkish coffee from different markets in Turkey.Method: Three samples were collected from each of teas, coffees, Turkish coffee, soft drinksand energy drinks at beverages sold different brands and forms. After maintainingstandardization in the preparation of beverages, the amounts of caffeine were analyzed withultraviolet determination method by HPLC.Results:The highest concentration of caffeine in drinks in the measurement was found to bethe Turkish coffee (858mg/L). Caffeine levels were measured in normal tea 840 mg/L ,in solublecoffee 509 mg/L, in tea bags 218 mg/L, in coke 148 mg/L and in energy drinks 169 mg/L.Discussion and Conclusion: Caffeine containing drinks are consumed through the entire lifefrom childhood to senescence. Received moderate amounts of caffeine provide positive benefitssuch as increased motivation, alertness, self-feeling energetic. Furthermore gradually with theincrease of amount consumed caffeine cause unwanted symptoms such as headache,nervousness. Therefore the knowledge of amount caffeine at daily use drinks in a country isimportant for health of consumersÖğe Dermal absorption and toxicity of alpha amanitin in mice(Informa Healthcare, 2014) Kaya, Ertuğrul; Sürmen, Mustafa Gani; Yaykaşlı, Kürşat Oğuz; Karahan, Selim; Oktay, Murat; Turan, Hakan; Erdem, HavvaThe fungus Amanita phalloides is known to contain two main groups of toxins: amanitins and phallotoxins. The amanitins group effectively blocks the RNA polymerase II enzyme found in eukaryotic cells. As alpha amanitin has a lethal effect on the majority of eukaryotic cells, it can be valuable as an antiparasitic or antifungal drug. It can be used externally against ectoparasites. It is critical that percutaneous applications of the alpha amanitin toxin are not harmful to the recipient. In this study, the absorption and the toxicity of percutaneous and intraperitoneal (ip) applications of 1 mg/kg alpha amanitin to mice were compared. Potential skin, liver and kidney toxicities were investigated through pathological examination. HPLC analysis was used to determine the amount of the toxin. No toxicity or toxin were found in the skin, liver, or kidneys of the mice in the control group. Interestingly, the percutaneous application group also showed no toxicity, and the toxin was not present in this group. After 24 h, Councilman-like bodies and pyknotic cells were observed in the mice in which alpha amanitin was applied intraperitoneally, demonstrating the presence of toxicity. Peak levels of alpha amanitin (mu g/mL) in the liver, kidney, and blood in the ip application group were measured at 3.3 (6 h), 0.2 (6 h) and 1.2 (1 h), respectively. The results demonstrated that the toxin was not absorbed through the skin of the mice and that the percutaneous application of alpha amanitin did not have any toxic effects. Thus, alpha amanitin may be administered percutaneously for therapeutic purposes.Öğe Düzce Yöresinde Yetişen Amanita Phalloides Mantarındaki Alfa Amanitin Düzeyinin Hplc Yöntemiyle Ölçümü(2012) Kaya, Ertuğrul; Karahan, Selim; Hancı, Mustafa; Yaykaşlı, Kürşat Oğuz; Sarıtaş, Ayhan; Bayram, Recep; Arslan, Seyfullah OktayAmaç: Düzce ili sınırlarında 2010 yılında toplanan Amanita phalloides mantarındaki alfa amanitin toksin düzeyinin HPLC yöntemiyle ölçümü amaçlanmıştır. Yöntem: Bir mantar bütün olarak, diğeri ise parçalara ayrılarak ekstraksiyon yapılmıştır. Ölçümler HPLC cihazında 303 nm UV dalga boyu, 250x4,6 mm C18 5 µm partikül içeren kolon kullanılarak gerçekleştirilmiştir. Mobil faz olarak amonyum asetat metanol asetonitril (801010, v/v/v) kullanılmış ve akış hızı 1 mL/dakikaya ayarlanmıştır. Sonuçlar 1 g kuru mantardaki toksin miktarı olarak verilmiştir. Bulgular: Bütün mantardaki alfa amanitin miktarı 4,806 mg (0,033), şapkada 3,522 mg (0,024), lamelde 5,318 mg (0,056), halkada 0,903 mg (0,004), sapta 2,577 mg (0,037), kapçıkta 0,698 mg (0,008) olarak ölçüldü. Sonuç: Düzce yöresinde yetişen Amanita phalloides mantarlarındaki alfa amanitin düzeyleri, başka bölgelerde yetişenlerden farklılık göstermektedir. Bulduğumuz sonuçlardan daha yüksek ve daha düşük seviyede toksin düzeyi ölçülmüş araştırmalar literatürde mevcuttur. Bu farklılığın etkenleri arasında iklim şartları yanında ekstraksiyon ve analiz yöntemlerindeki farklılıklar da rol oynayabilir.Öğe Gene expression profiles for apoptotic and necrotic pathways during Amanita phalloides intoxication in mice(2022) Karahan, Selim; Atlı, Zehra; Kaya, Ertuğrul; Özdemir, Feride; Boğa, Mehmet; İzgi, SevcanBackground and Aims: Amanita phalloides is the deadliest toxic mushroom in the world and causes death from acute liver failure. ?-amanitin (?-AMA), the most potent toxin, inhibits RNA polymerase II in hepatocytes, stops protein synthesis, and causes hepatotoxicity. However, the information about the mechanisms underlying hepatotoxicity caused by ?-AMA is quite inadequate. This study aims to reveal the complex necrotic and apoptotic mechanisms occurring in mouse hepatocytes de- pending on A. phalloides exposure time in vivo. Methods: BALB-c male mice were divided into 5 groups (n=7): control, ?-AMA-2, ?-AMA-12, ?-AMA-72, and ?-AMA-96 groups. A poisoning model was created by oral administration of A. phalloides mushroom extract containing 10 mg/kg of ?-AMA to mice and they were sacrificed after 2, 12, 72, and 96 h. Then, TNF-?, Bax, caspase-3, and Bcl-2 gene expression levels in liver tissues were examined by the RT-qPCR method. Time-dependent damage to liver tissues was also evaluated histopathologically. Results: RT-qPCR results showed that proinflammatory cytokine TNF-? mRNA expression levels increased in mouse liver tissues at 2 and 12 h after A. phalloides administration compared among the groups. Bax mRNA expression levels increased in the 12 and 72 h after A. phalloides ingestion. It was observed that caspase-3 mRNA expression levels increased in the 72 and 96 h groups compared among the groups, while Bcl-2 mRNA expression levels decreased in the 72 and 96 h groups. Conclusion: Our findings showed that necrotic mechanisms develop in the early period after A. phalloides mushroom poison- ing, and then apoptotic mechanisms are effective. In conclusion, understanding the mechanisms of A. phalloides-induced hepatotoxicity will provide important information for new treatment strategies to be developed.Öğe Gene expression profiles for apoptotic and necrotic pathways during Amanita phalloides intoxication in mice(Istanbul Univ, Fac Pharmacy, 2022) Karahan, Selim; Atli, Zehra; Kaya, Ertugrul; Ozdemir, Feride; Boga, Mehmet; Izgi, SevcanBackground and Aims: Amanita phalloides is the deadliest toxic mushroom in the world and causes death from acute liver failure. alpha-amanitin (alpha-AMA), the most potent toxin, inhibits RNA polymerase II in hepatocytes, stops protein synthesis, and causes hepatotoxicity. However, the information about the mechanisms underlying hepatotoxicity caused by alpha-AMA is quite inadequate. This study aims to reveal the complex necrotic and apoptotic mechanisms occurring in mouse hepatocytes de-pending on A. phalloides exposure time in vivo.Methods: BALB-c male mice were divided into 5 groups (n=7): control, alpha-AMA-2, alpha-AMA-12, alpha-AMA-72, and alpha-AMA-96 groups. A poisoning model was created by oral administration of A. phalloides mushroom extract containing 10 mg/kg of alpha-AMA to mice and they were sacrificed after 2, 12, 72, and 96 h. Then, TNF-alpha, Bax, caspase-3, and Bcl-2 gene expression levels in liver tissues were examined by the RT-qPCR method. Time-dependent damage to liver tissues was also evaluated histopathologically.Results: RT-qPCR results showed that proinflammatory cytokine TNF-alpha mRNA expression levels increased in mouse liver tissues at 2 and 12 h afterA. phalloides administration compared among the groups. BaxmRNA expression levels increased in the 12 and 72 h afterA. phalloides ingestion. It was observed that caspase-3 mRNA expression levels increased in the 72 and 96 h groups compared among the groups, while Bcl-2 mRNA expression levels decreased in the 72 and 96 h groups.Conclusion: Our findings showed that necrotic mechanisms develop in the early period afterA. phalloides mushroom poison-ing, and then apoptotic mechanisms are effective. In conclusion, understanding the mechanisms of A. phalloides-induced hepatotoxicity will provide important information for new treatment strategies to be developed.Öğe The measurement of alpha amanitin levels using hplc method in Amanita phalloides from Düzce province(Duzce University Medical School, 2012) Kaya, Ertuğrul; Karahan, Selim; Hancı, Mustafa; Yaykaşlı, Kürşat Oğuz; Sarıtaş, Ayhan; Bayram, Recep; Arslan, Seyfullah OktayAim: The aim of this study is to measure the level of alpha amanitin toxin using HPLC method from Amanita phalloides mushroom collected in the province of Düzce in 2010. Method: One of the mushrooms as a whole body. The other one was extracted after seperated into parts. The measurement was done by HPLC using 303 nm UV wavelength and 250x4,6 mm C18 5 ?m particle included column. Ammonium acetate+methanol+acetonitrile (80+10+10, v/v/v) was used as a mobile phase, and the flow rate was set 1 ml/min. The results were given as a toxin quantity in 1 g dry mushroom. Results: The amount of alpha amanitin was measured as 4,806 mg (±0,033) in the whole body, 3,522 mg (±0,024) in the cap, 5,318 mg (±0,056) in the lamellar, 0,903 mg (±0,004) in the ring, 2,577 mg (±0,037) in the stipe, 0,698 mg (±0,008) in the volva. Conclusion: The level of alpha amanitin in Amanita phalloides from Duzce Province is differ from different countries. Higher and lower levels of toxin than our data obtained investigations are present in the literature. The reason of this differences might be several factors like extraction methods, analysis methods and environmental conditions. © 2012 Düzce Medical Journal.Öğe Yüksek Saflıkta Beta Amanitin Üretimi(2012) Kaya, Ertuğrul; Yaykaşlı, Kürşat Oğuz; Karahan, Selim; Bayram, Recep; Sarıtaş, Ayhan; Yaykaşlı, EmineAmaç: Beta amanitin nadiren bilimsel araştırmalarda kullanılmakta ve kullanımı gün geçtikçe artmaktadır. Piyasada bu ürün %90 saflıkta ticari olarak satılmaktadır. Bu araştırmada yüksek saflıkta beta amanitin üretme yöntemi tanımlanmıştır. Yöntem: Saflaştırma işlemi Amanita phalloides mantarlarından ekstraksiyonla yapılmıştır. Öncelikle bu mantarlar toplanmış, ekstrakte edilmiş, 2 defa preparatif HPLC ile saflaştırma işlemi uygulanmıştır. Toksinin karşılaştırması, analitik HPLC sisteminde tutulma zamanı ve ultraviyole spektrumu karşılaştırılması yöntemleriyle yapılmıştır. Bulgular: İlk saflaştırma sonucunda elde edilen beta amanitin saflık oranı %91(2,36) olarak ölçülmüştür. İkinci saflaştırma sonucunda elde edilen beta amanitin saflık oranı %99,2(0,38) olarak ölçülmüştür. Saflaştırma sonucu elde ettiğimiz toksin ile beta amanitin standardın UV spektrumlarında her ikisinde de 303 nmde maksimum, 263 nmde minimum absorbans verdiği ve spektrum yapısının aynı olduğu görülmüştür. Sonuç: Tanımladığımız bu yöntemle, %99 saflıkta beta amanitinin üretilmesi mümkündür.
