المؤلفون: Magdalan, Jan, Piotrowska, Aleksandra, GomuŁkiewicz, Agnieszka, Sozański, Tomasz, Podhorska-OkoŁów, Marzena, Szeląg, Adam, Dzięgiel, Piotr

المصدر: Experimental & Toxicologic Pathology; May2011, Vol. 63 Issue 4, p311-315, 5p

مصطلحات موضوعية: PENICILLIN, AMANITINS, APOPTOSIS, LIVER cells, AMANITA phalloides, RNA polymerases, PROTEIN synthesis, DNA synthesis, CELL culture

مستخلص: Abstract: High mortality rate in Amanita phalloides (death cap) intoxications is a result of the acute liver failure following hepatocyte damage due to hepatocellular uptake of amatoxins. α-Amanitin (α-AMA), the major amatoxin, blocks a RNA polymerase II, which results in inhibition of transcription of DNA and protein synthesis processes and leads to hepatocyte death. α-AMA is also a strong apoptosis inductor and may play a significant role in pathogenesis of hepatic damage in course of amanitin intoxication. The aim of this study was to examine mechanisms of α-AMA-induced apoptosis in human hepatocytes, as well as in determining if commonly clinically used antidotes benzylpenicillin (BPCN) and N-acetylcysteine (ACC) are able to protect human hepatocytes against α-AMA-induced apoptosis. The experiment was performed on cultured human hepatocytes. Viability of cultured hepatocytes was assessed using the MTT assay, whereas apoptosis processes were evaluated by the electron microscopy, detection of DNA laddering, determination of caspase-3 activity, and measuring annexin V, p53 and Bcl-2 protein concentration. Cytotoxicity and apoptosis evaluation were performed after 24h of exposure to α-AMA and/or tested antidotes.Both ACC and BPCN were well tolerated by human hepatocyte cultures, and exposure to those substances did not reduce cell viability nor induce apoptosis. Exposure of hepatocytes to α-AMA at concentration 2μM resulted in derangement of cell cultures, apoptosis and significant reduction in cell viability. α-AMA-induced apoptosis in human heptocyte cultures is p53- and caspase-3-dependent. Human hepatocyte cultures are exposed simultaneously to α-AMA and tested antidotes (BPCN or ACC) showed significantly higher cell viability and significantly lower values of apoptosis markers compared to the cultures exposed to α-AMA only. [Copyright &y& Elsevier]

: Copyright of Experimental & Toxicologic Pathology is the property of Urban & Fischer Verlag and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

المؤلفون: Magdalan, Jan, Ostrowska, Alina, Piotrowska, Aleksandra, Gomułkiewicz, Agnieszka, Podhorska-Okołów, Marzena, Patrzałek, Dariusz, Szeląg, Adam, Dzięgiel, Piotr

المصدر: Experimental & Toxicologic Pathology; Jul2010, Vol. 62 Issue 4, p367-373, 7p

مصطلحات موضوعية: PENICILLIN, ANTIDOTES, LIVER cells, FOOD poisoning, AMANITINS, AMANITA phalloides, CELL-mediated cytotoxicity, CELL proliferation

مستخلص: Abstract: Fatalities due to mushroom poisonings are increasing worldwide, with high mortality rate resulting from ingestion of amanitin-producing species. Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology since no specific and fully efficient antidote is available. The objective of this study was a comparative evaluation of benzylpenicillin (BPCN), acetylcysteine (ACC) and silibinin (SIL) as an antidotes in human hepatocytes intoxicated with α-amanitin (α-AMA). All experiments were performed on cultured human hepatocytes. Cytotoxicity evaluation of cultured cells using MTT assay and measurement of lactate dehydrogenase (LDH) activity was performed at 12, 24 and 48h of exposure to α-AMA and/or antidotes. The significant decline of cell viability and significant increase of LDH activity were observed in all experimental hepatocyte cultures after 12, 24 and 36h exposure to α-AMA at concentration 2μM. Exposure of the cells to α-AMA resulted also in significant reduction of cell spreading and attachment. However, addition of tested antidotes to experimental cultures significantly stimulated cell proliferation and attachment. In cell cultures exposed simultaneously to α-AMA and tested antidotes cytotoxic parameters (MTT and LDH) were not significantly different from control incidences. The cytoprotective effect of all antidotes was not dose-related, which reflects a high efficacy of all these substances. Administration of studied antidotes was not associated with any adverse effects in hepatocytes. The administration of ACC, BPCN or SIL to human hepatocyte cultures showed a similar strong protective effect against cell damage in α-AMA toxicity. [Copyright &y& Elsevier]

: Copyright of Experimental & Toxicologic Pathology is the property of Urban & Fischer Verlag and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

المؤلفون: Magdalan, Jan, Ostrowska, Alina, Piotrowska, Aleksandra, Iżykowska, Ilona, Nowak, Marcin, Gomułkiewicz, Agnieszka, Podhorska-Okołów, Marzena, Szeląg, Adam, Dzięgiel, Piotr

المصدر: Folia Histochemica et Cytobiologica; 2010, Vol. 48 Issue 1, p58-62, 5p

مصطلحات موضوعية: AMANITINS, APOPTOSIS, CELL death, LIVER cells, LABORATORY dogs

مستخلص: Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. Awide variety of amatoxins have been isolated; however, α-amanitin (α-AMA) appears to be the primary toxin. Studies in vitro and in vivo suggest that α-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in α-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with α-AMA at a final concentration of 1, 5, 10 and 20 μM. Viability test (MTT assay), apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy) were performed at 6 and 12 h of exposure to α-AMA. There was a clear correlation between hepatocyte viability, concentration of α-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to α-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning. [ABSTRACT FROM AUTHOR]

: Copyright of Folia Histochemica et Cytobiologica is the property of VM Medica-VM Group (Via Medica) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

المؤلفون: Magdalan, Jan¹, Ostrowska, Alina¹ al•ostra@yahoo.com, Piotrowska, Aleksandra², Gomułkiewicz, Agnieszka², Szeląg, Adam¹, Dzięgiel, Piotr^2,3

المصدر: Archives of Toxicology. Dec2009, Vol. 83 Issue 12, p1091-1096. 6p. 2 Charts, 4 Graphs.

مصطلحات موضوعية: *RIFAMYCINS, *LIVER cells, *AMANITINS, *CELL culture, *DEHYDROGENASES

مستخلص: The most often used antidote to treat poisoning caused by amanitin-containing mushrooms is benzylpenicillin (BPCN). However, a very few reports suggest that other antibiotics such as ceftazidime (CEFT) and rifamycin SV (RIFSV) show better antidote activity against amanitins than BPCN. Given this, there is an ongoing debate as which of three antidotes is optimal for treatment of such poisonings. In this study, the efficacy of BPCN was compared with those of CEFT and RIFSV in human hepatocyte model. The functional integrity and viability of cultured hepatocytes was evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and by measurements of lactic dehydrogenase (LDH) activity. In the first experimental layout, hepatocytes were simultaneously exposed to α-AMA and tested antidotes, while in the second layout, the cells were exposed for the first 12 h to α-AMA only, and then, the medium containing α-AMA was exchanged to culture medium containing both α-AMA and the antidotes tested. The results demonstrated that simultaneous administration of α-AMA and each of tested antidotes (BPCN, CEFT, RIFSV) effectively protected human hepatocytes; however, in the group dosed with BPCN, the highest hepatocyte viability was observed. In cell cultures from experimental layout II, all tested antidotes were ineffective, which indicates that after the critical dose of α-AMA had been taken up by hepatocytes, further suppression of this process does not protect the cells against injury. Thus, 12 h of exposure of incubated hepatocytes to α-AMA is a sufficient time for such a cellular uptake of a critical dose of this toxin. In summary, it can be concluded that easily accessible and low-cost BPCN should be widely used as an antidote against amanitins. However, the key to successful therapy is a quick implementation of an antidote in order to protect as large as possible portion of the liver parenchyma against the devastating uptake of a critical dose of amanitins. [ABSTRACT FROM AUTHOR]

المؤلفون: Magdalan, Jan¹, Ostrowska, Alina² al_ostra@yahoo.com, Podhorska-Okołów, Marzena³, Piotrowska, Aleksandra³, Iżykowska, Ilona³, Nowak, Marcin⁴, Dolińska-Krajewska, Barbara³, Zabel, Maciej^3,5, Szeląg, Adam¹, Dzięgiel, Piotr³

المصدر: Archives of Toxicology. Jan2009, Vol. 83 Issue 1, p55-60. 6p. 4 Diagrams, 4 Graphs.

مصطلحات موضوعية: *AMANITA phalloides, *LIVER cells, *MUSHROOM poisoning, *AMANITINS, *LACTATE dehydrogenase, *TOXINS

مستخلص: The toadstool death cap ( Amanita phalloides) and its subspecies, destroying angel ( A. virosa) and death angel ( A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of α-amanitin (α-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of α-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure. [ABSTRACT FROM AUTHOR]