Tesi sul tema "Cyclopiazonic acid"

O objetivo deste estudo foi avaliar a presença de fungos e micotoxinas [aflatoxinas e ácido ciclopiazônico (ACP)] em amostras de castanha-do-Brasil coletadas em diferentes estados da região Amazônia brasileira: Acre, Amazonas, Amapá e Pará. Um total de 200 amostras de casca e 200 amostras de amêndoa foram semeadas em ágar Aspergillus flavus-parasiticus para pesquisa da micobiota. As micotoxinas foram analisadas por cromatografia líquida de alta eficiência. A morfológica clássica e a análise molecular revelou a presença dos seguintes fungos, em ordem decrescente de frequência: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. e Aspergillus spp. A presença de aflatoxinas e ACP foi detectada em amostras de amêndoa, na frequência de 11% e 22%, respectivamente. Aspergillus nomius e Aspergillus flavus foram as espécies mais isoladas de Aspergillus. A presença de fungos não implica necessariamente na contaminação por micotoxinas, porém a amêndoa da castanha-do-Brasil parece ser um bom substrato para o crescimento de fungos.
The aim of this study was to evaluate the presence of fungi and mycotoxins [aflatoxin and cyclopiazonic acid (ACP)] in Brazil nut samples collected in different States of the Brazilian Amazon: Acre , Amazonas , Amapá and Pará. A total of 200 almond and 200 husk samples were plated on agar Aspergillus flavus - parasiticus for mycobiota isolation. Mycotoxins were analyzed by high performance liquid chromatography. The classical morphological and molecular analysis revealed the presence of the following fungi , in decreasing order of frequency: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. and Aspergillus spp. The presence of aflatoxins and ACP was detected in almonds samples at a frequency of 11% and 22% , respectively. Aspergillus nomius and Aspergillus flavus species were the most isolated from Aspergillus. The presence of fungi does not necessarily imply the mycotoxin contamination, but the almond samples of Brazil nut seem to be a good substrate for the growth of fungi.

Submitted in partial fulfillment of the requirements for the degree of Masters in Technology in Chemistry, M.L. Sultan Technikon, 2000.
Mycotoxins are a group of natural poisons produced by certain strains of fungal species when they grow under favourable conditions on a wide variety of different substrates. These toxins have been implicated in a wide range of acute diseases in man and animals. Their toxic effects include oesophageal cancer and liver diseases in humans, and carcinogenic effects in experimental rats and poultry. Hence, there is a need to monitor toxin levels in food commodities.
M

碩士
國防醫學院
藥理學研究所
89
1. Aims: (1) To investigate the role of the sarcoplasmic reticulum ( SR ) in the abnormal force development of the Syrian hamster model in late stage of cardiomyopathy (39~43 week-old). (2) We studied the role of SR by evaluating the change in twitch force after a rest interval as the change in force is believed to be a function of Ca reuptake into the terminal cisternae of the SR. Furthermore, we used a SR Ca2+ reuptake inhibitor ( cyclopiazonic acid, CPA ) to unmask an abnormal Ca2+ uptake by the SR. (3) To evaluate whether and to what extent the results obtained in our animal model applies to human tissue, we also studied ventricular tissue from human myopathic heart ( idiopathic dilated myopathy ) under the same conditions as ventricular myocardium of hamster to compare and contrast the abnormality of function. 2. Methods: (1) Three groups of Syrian hamsters were used in the present study: 12 male myopathic hamsters ( strain Bio 14.6 ) at age of 16 weeks were obtained from the Biobreeders Company ( USA ) and raised in the animal house of our medical center until age of 39~43 weeks. Eight age-matched healthy hamsters ( strain F1B ) were obtained and raised in the same manner and served as control. In addition, 8 healthy Syrian hamsters were obtained locally from the National Science Coucil Animal Center for comparative study. (2) Papillary muscle ( diameter around 1.5 mm ) obtained from left ventricle and perfused in vitro at 37 oC. Action potentials ( APs ) were recorded with the conventional microelectrode techniques. Twitch and diastolic force were recorded by means of a force-displacement transducer. In an additional series of experiments, hamster hearts were perfused in a Langendorff apparatus with perfusate containing digesting enzymes. Single cardiomyoctes were isolated and ionic currents were determined with the whole-cell patch-clamp techniques at room temperature. (3) Human ventricular muscles obtained from the right ventricles of 8 patients with ideopathic dilated cardiomyopathy (7 male 1 female, age 37 ± 7 years) underwent heart transplantation were studied in a similar manner. 3. Results and discussion: (1) Steady-state twitch force and PRPC after a rest interval The relation between PRPC and the ability of SR to take up cytoplasmic Ca2+ was tested in hamster driven at a basic rate of 2 Hz. When the stimulation was interrupted for 2 ~ 60 s, the first post-rest twitch force was markedly increased with respect to that prior to the pause. Also, the second twitch curve was still larger than control and it took several beats for force to go back to control value. Also in the human tissue driven at a basic rate of 1 Hz, after the 2 ~ 60 s pause the twich force was larger than prior to the pause. However, not only the increase in force was less than in myopathic hamster, but the potentiation had already subsided by the second twitch after the pause. (2) Force development at increasing driving rates When the active and passive force development was recorded while the driving rate was increased from 0.5 Hz to 1, 2 and 3 Hz, the twitch force became larger as driving rate was increased, consistent with an increased intracellular Ca2+ and therefore an increased Ca2+ uptake into the SR. However, at 3 Hz the diastolic tension did not relax completely. After the pause, there was a PRPC only for the first beat. When the same procedure was repeated in the presence of CPA, the contraction was smaller. But as Ca2+ load was increased at 3 Hz, there was a marked increase in diastolic force, as the further limitation of the uptake of Ca into the SR by CPA resulted in a larger increase cytoplasmic diastolic Ca2+. During the pause, contracture subsided quickly. Consistent with a reduced uptake of Ca2+ by the SR, first post-rest twitch force was markedly reduced. (3) Post-rest potentiation of contraction (PRPC) and CPA at two different rates PRPC was studied in human ventricular tissue at 1 and 3 Hz to compare and contrast the effect of a different Ca2+ load. The faster rate resulted in a smaller systolic tension and an increase in diastolic tension compared to the slower rate. However, during the pause the diastolic tension quickly decreased to the baseline, which suggests that Ca2+ was moved from the cytoplasm into the SR. This appears to be confirmed by the fact that at 3 Hz PRPC was larger and also by the fact that in the presence of CPA (10 µM) the resting tension decreased far more slowly and PRPC was almost eliminated. Also the recovery from CPA effects was rather slow and incomplete. (4) Influences of rest interval on PRPC PRPC was studied as a function of duration of the rest interval (2, 5, 10, 20, 40 and 60 s) in ventricular myocardium of 39~43 week-old healthy and myopathic hamsters. The general shape of the PRPC-rest interval relationship was similar to that of the younger hamsters (17~27 week-old) in our previous study, but the curve of the older myopahtic ventricular myocardium was obviously shifted downward. CPA decreased predominantly the ascending part of the curve in both the healthy and the myopathic myocardium. When the PRPC-rest interval relations were tested in human ventricular myocarium. The curve of PRPC-rest interval peaked at longer interval (40~60 s) compared to that of the hamsters. Another difference is that CPA markedly depresses the relation throughout the curve. Therefore the behavior of human tissue again resemble that of the older myopathic hamster, as it might be expected from the fact that the disease is likely to have reached a more advanced stage in human tissues. The fact that in the human ventricular myocardium the relation falls but little at the longer intervals might be due to the fact that Ca extrusion is more compromised (and the cytoplasmic [Ca2+] higher). Also, the marked decrease of the PRPC at all the rest intervals indicates that Ca2+ uptake into the SR may be more markedly affected than in the older myopathic hamster model. (5) APD near zero potential (APD0mV) was markedly shortened during the first post-rest beat, especially after CPA (10 µM) exposure in healthy hamster, indicating complete recovery of transient outward K currents (Ito) after a rest interval of 20 s and longer. Post-rest shortening in APD0mV was smaller in myopathic hamster. Measurements of ionic currents under voltage-clamp show that the healthy hamster ventricular myocytes have a very strong Ito on depolarization. CPA consistently enhanced the Ito in all 7 myocytes tested. (6) APD90 was significantly prolonged after a rest interval along with the greatly increased post-rest contraction, especially in hamster ventricular myocardium. It is assumed that an increased extrusion of Ca2+ through the Na+/Ca2+ exchanger could be the underlying ionic mechanism. (7) After prolonged depolarizing step (from —40 to +40 mV, > 3 s), transient inward currents (Iti) were induced on repolarization in 6 of 7 hamster cardiomyocytes tested. CPA (3 μM) suppressed irreversibly the Iti in 2 myocytes but enhanced reversibly the Iti in other 4 myocytes. A depletion of SR Ca2+ stores and an enhanced release of Ca2+ from the SR, respectively, are assumed to be the underlying causes for these 2 seemingly opposite actions. 4. Conclusions: (1) The present results show that in ventricular myocardium of patients affected by dilated cardiomyopathy, mechanical behavior shows similarity and differences with the myopathic Syrian hamster model. (2) The similarities include PRPC with a distinctive dependence on driving rate and the depression of PRPC after inhibition of Ca2+ uptake into the SR by CPA. The differences include the development of diastolic tension at faster rates (especially in the presence of CPA) and the more marked depression of this relation by CPA. Therefore, the present results show there are pecularities that human myopathic ventricular tissue does not share with the hamster model and that might indicate a more serious deterioration of function in ventricular myocardium presumably because patient underwent cardiac transplantation when the disease had reached the stage of cardiac failure. (3) The hamster ventricular myocytes have a very strong transient outward K currents (Ito) on depolarization, in agreement with a rather narrow APD near zero potential. CPA consistently enhanced the Ito but could depress or enhance the oscillatory transient inward currents (Iti) on repolarization. Changes in Iti may explain the suppression or genesis of arrhythmis during or after CPA exposure. Further experiments are required to clarify the differences between the electrophysiological properties of myopathic vs. healthy hamsters and between human and hamster cardiomyocytes. (4) A more general conclusion is that, while animal study provide interesting insight for the understanding of human disease, only studies in human tissues provide direct evidence for the abnormalities of function caused by disease.

Tese de doutoramento programa doutoral em Engenharia Química e Biológica
The reduction of yield, quality, and nutritional value of grain cereals by filamentous fungi and subsequent contamination with mycotoxins is of great concern around the world. Mycotoxins are known to cause serious health problems in animals and some mycotoxins such as aflatoxins, fumonisins and ochratoxins, in particular, have also been associated with human health problems. Aflatoxins, fumonisins and ochratoxin A produced by several Aspergillus species are prominent among the mycotoxins associated to maize economic losses (Zea mays L.). However, the presence of a given fungus does not mean that the mycotoxin(s) associated with that fungus is (are) also present. There are many factors, especially environmental conditions and agricultural practices, involved in the production of mycotoxins. The aim of this thesis was to contribute to the risk assessment of post-harvested contamination of maize kernels with aflatoxins (AFs), cyclopiazonic acid (CPA), fumonisins B1 and B2 (FB1 and FB2, respectively) and ochratoxin A (OTA) in three Portuguese regions (Beira Litoral, Ribatejo e Alto Alentejo). To this end, studies were made, first to determine maize Aspergillus mycoflora and assess the mycotoxigenic potential of the isolated strains, and secondly to determine the incidence of AFs, CPA, OTA and FB in milled maize. Ninety five maize samples were collected between November 2008 and April 2009 in maize association of producer’s facilities in three different agroclimatic regions of Portugal. These samples were taken at reception, after drying and after storage, and moisture content was measured immediately after sampling. Strains of Aspergillus were isolated and identified through phenotypic characterization and mycotoxin production. Molecular work was also done in eight isolates that could not be fully identified, up to species level, and a new species belonging to Aspergillus section Flavi was described and named as Aspergillus mottae Around ninety one percent of the maize samples were contaminated with strains of Aspergillus. These were subsequently grouped into five sections: Flavi (423 isolates) followed by Nigri (270 isolates), Wentii (214 isolates), Circumdati (15 isolates) and Candidus (2 isolates). The fungal frequency of the isolations obtained from the samples belonging to the three steps of the maize storage chain differs. The highest frequency occurs after drying, being the lowest frequency at reception followed by storage. What concerns Aspergillus species isolations at each region, the regions with Mediterranean climates had a higher percentage of Aspergillus section Nigri isolates in comparison with the region with transitional between Atlantic and Mediterranean climate. Maize samples were analyzed by HPLC for mycotoxin contamination. Mycotoxins were detected in 83% of the samples, with prevalence for FB1. Hence, 83% percent of the samples were positive for FB1, 46% were positive for FB2 and only 5% were positive for AFs. None of the samples were positive for OTA and CPA. In conclusion, the two Producer’s Society and the Agricultural Cooperative showed to have good practices, minimizing the occurrence of mycotoxins. Even though Aspergillus section Flavi, especially of A. flavus, and Aspergillus section Nigri isolates were present in high numbers in the maize kernels, very few samples were contaminated with aflatoxins (5%), and none were positive for either CPA and OTA. The presence of fumonisins (FB1 and FB2) in the majority of the samples (83 and 46%, respectively) suggests that there was pre-harvest contamination with Fusarium species. There is no evidence that the presence of FB2 is due to the presence of Aspergillus niger aggregate isolates.
A redução do rendimento, qualidade e valor nutricional dos cereais por fungos filamentosos e consequente contaminação com micotoxinas é de grande preocupação em todo o mundo. As micotoxinas são conhecidos por causar sérios problemas de saúde nos animais e algumas micotoxinas em particular as aflatoxinas, fumonisinas e ocratoxinas, também têm sido associadas a problemas de saúde nos seres humanos. As aflatoxinas, fumonisinas e ocratoxina A, produzidas por várias espécies de Aspergillus, estão entre as micotoxinas associadas a perdas económicas no milho (Zea mays L.). No entanto, a presença de um dado fungo não significa que a(s) micotoxina(s) associada também esteja(m) presente. Há muitos factores envolvidos na produção de micotoxinas, tendo especial relevância as condições ambientais e as práticas agrícolas. O objectivo desta tese foi contribuir para a avaliação do risco de contaminação póscolheita de grãos de milho com aflatoxinas (AFs), ácido ciclopiazónico (CPA), fumonisinas B1 e B2 (FB1 e FB2, respectivamente) e de ocratoxina A (OTA), em três regiões portuguesas (Beira Litoral, Ribatejo e Alto Alentejo). Para este fim, foram realizados estudos com o intuito de determinar, em primeiro lugar, a variedade de Aspergillus associada ao milho e o potencial micotoxigénico das estirpes isoladas, e, em segundo lugar, determinar a incidência de AFs, CPA, OTA e FB em farinha de milho. De modo a atingir os objectivos, foram recolhidas noventa e cinco amostras de milho entre Novembro de 2008 e Abril de 2009 em Associações de Produtores de milho situadas em três regiões de Portugal com diferenças agroclimáticas. Estas amostras foram retiradas na recepção, após a secagem e após o armazenamento, tendo sido medido o teor de humidade logo após a amostragem. As estirpes de Aspergillus foram isoladas e identificadas através da caracterização fenotípica e da produção de micotoxinas. Foram realizados estudos moleculares em oito isolamentos, uma vez que não puderam ser identificados até ao nível de espécie. O resultado deste trabalho permitiu descrever uma nova espécie pertencente aos Aspergillus secção Flavi, cuja designação passou a ser Aspergillus mottae. À volta de de 91% das amostras de milho estavam contaminadas com estirpes de Aspergillus. As estirpes foram identificadas como pertencentes a cinco diferentes secções: Flavi (423 isolamentos), seguida por Nigri (270 isolamentos), Wentii (214 isolamentos), Circumdati (15 isolamentos) e Candidus (2 isolamentos). A frequência fúngica entre os isolamentos obtidos a partir das amostras pertencentes às três etapas da cadeia de armazenamento de milho difere. A maior frequência ocorre após a secagem, sendo a menor frequência na recepção seguida do armazenamento. No que refere a isolamentos de espécies de Aspergillus em cada região, as regiões com climas Mediterrânicos apresentaram maior percentagem de isolamentos de Aspergillus secção Nigri em comparação com a região de clima de transição entre Atlântico e Mediterrânico. As amostras de milho recolhidas foram analisadas por HPLC para determinar se estariam contaminadas com micotoxinas. As micotoxinas foram detectadas em 83% das amostras, com prevalência para FB1. Assim, 83% por cento das amostras foram positivas para FB1, 46% foram positivas para FB2 e apenas 5% foram positivas para AFs. Nenhuma das amostras acusou a presença de OTA e CPA. Em conclusão, as Associações de Produtores mostraram ter boas práticas, minimizando a ocorrência de micotoxinas. Mesmo que a presença de estirpes de Aspergillus secção Flavi, principalmente de A. flavus, e de estirpes de Aspergillus secção Nigri estivessem presentes em números elevados nos grãos de milho, poucas amostras mostraram estar contaminadas com aflatoxinas (5%), e nenhum delas foi positiva quer para CPA quer para OTA. A presença de fumonisinas (FB1 e FB2) na maioria das amostras (83 e 46%, respectivamente) sugere que houve uma contaminação pré-colheita com espécies de Fusarium. Não há nenhuma prova que indique que a presença de FB2 é devido à presença de estirpes pertencentes ao agregado Aspergillus niger