First report of anatoxin-a encoding gene in isolated cyanobacterial strains from Malaysia

@#Aims:This study focus on the presence of cyanobacterial toxin in Malaysia and anatoxin-a-encoding gene was detected in this study and the status of cyanobacterial toxins in Malaysia can now be clarified.Methodology and results:As part of status determination of cyanobacterial toxins in Malaysia, cyanobacterial strains have been isolated from different environments and identified using cyanobacterial16S rRNA gene sequence. PCR assay was carried out to detect the presence of cyanobacterial toxin-encoding genes in these isolated strains by amplifying genes encoded for microcystin, anatoxin-a, cylindrospermopsin and saxitoxin. Using molecular identification of 16S rRNA gene sequences, a total of forty-two cyanobacterial strains were identified, which belongs to eighteen different genera of Synechococcus, Cyanobium, Synechocystis, Chroococcidiopsis, Leptolyngbya, Nodosilinea, Limnothrix, Pseudanabaena, Cephalothrix, Aerosakkonema, Oscillatoria, Alkalinema, Pantanalinema, Planktolyngbya, Scytonema, Nostoc, Hapalosiphonand Symphyonemopsis. The toxicity of these strains was tested using PCR amplification of toxin-encoding genes using specific primers.Conclusion, significance and impact of study:Anatoxin-a (ATX) gene,which involved in the biosynthesis of anatoxin-Awas detected in two isolated strains namelyLimnothrixsp. B15 and Leptolyngbyasp. D1C10.This study focus on the the presence of cyanobacterial toxin in Malaysia can now be determined as potential threat because anatoxin-a-encoding gene was detected in this study and the status of cyanobacterial toxins in Malaysia can now be clarified.

Effects of a cyanobacterial extract containing-anatoxin-a(s) on the cardiac rhythm of Leurolestes circunvagans

This work presents the effects of an anatoxin-a(s)-containing extract on a cockroach semi-isolated heart preparation and the results supporting the extract’s biological activity on acetylcholinesterase (purified from ell). The presence of the toxin in cyanobacterial strains Anabaena spiroides (ITEP-024, ITEP-025 and ITEP-026) isolated from the Tapacurá reservoir in Pernambuco, Brazil, was confirmed by means of liquid chromatography coupled to an ion-trap mass spectrometer. The anticholinesterase activity was assessed biochemically by the Ellman test and was confirmed by measuring the cockroach’s heart rate. The concentration of the extract containing the tested anatoxin-a(s) (antx-a(s)) (10, 16 and 100 μg.μL-1) inhibited the eel acetylcholinesterase (AChE) by more than 90%. The cockroach cardiac frequency increased by a maximum of about 20% within 29 min after the addition of 2.5x10³ μg of extract containing antxa (s).g-1 bw (n=9, p<0.05). Our results strongly indicate that antx-a(s) is capable of exerting biological effects on cockroach, indicating that more research might be conducted to determine its role in the environment, especially on insects.

Investigação de mecanismos bioquímicos e fisiológicos em organismos expostos a anatoxina-a(s) / Study of the biochemical and physiologycal mechanisms in organisms exposed to anatoxin-a(s)

A anatoxina-a(s) (antx-a(s)) é uma neurotoxina produzida por cianobactérias, cujo mecanismo de ação conhecido consiste na inibição irreversível da atividade da enzima acetilcolinesterase (AChE). Em razão da instabilidade da molécula e da inexistência de um padrão analítico, as informações referentes a presença dessa toxina nos reservatórios de água doce e, a completa elucidação dos processos envolvidos na sua toxicidade, são escassas. Na última década, a pesquisa referente a capacidade dos inseticidas organofosforados de induzir estresse oxidativo em humanos e animais tem sido bastante importante. Pelo fato da antx-a(s) ser o único organofosforado produzido por cianobactérias conhecido, este trabalho teve como objetivo investigar os mecanismos bioquímicos envolvidos na ação pró-oxidante de extratos aquosos contendo antx-a(s). A atividade das enzimas colinesterásicas AChE e butirilcolinesterase (BuChE) e, das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR) e superóxido dismutase (SOD) foram avaliadas em dois modelos experimentais: (i) Camundongos Swiss tratados por via intraperitoneal (ip) com uma dose sub-letal (20 mg.kg-1) de extrato contendo antx-a(s) e sacrificados após 24 h, 48 h, 7 e 14 dias e, (ii) sementes de alfafa regadas com extrato contendo antx-a(s) por um período de 7 dias. Também foi avaliado o efeito de extratos contendo antx-a(s) em coração de baratas da espécie Leurolestes circunvagans. A atividade enzimática das colinesterases avaliadas em camundongos tratados com extrato contendo antx-a(s) manteve-se inibida 58% até 48 h após tratamento (n=9, p<0,001), voltando a níveis normais (comparada ao controle) a partir do sétimo dia (n=10, p>0,05). Em contraposição, as alterações na atividade das enzimas antioxidantes avaliadas iniciaram-se após 48 h do tratamento, quando foi observada uma diminuição na atividade das enzimas CAT e GPx (n=9, p<0,001). No sétimo dia, enquanto a atividade das enzimas CAT...

Anatoxin-a(s) (antx-a(s)) is a cyanobacterial neurotoxin whose principal mechanism of action is the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AChE). Because of the molecule´s instability and the lack of an analytical standard, the occurrence of this toxin in freshwater reservoirs and the knowledge of the whole events related to its toxicity are scarce. In the last decade, research related to organophosphates insecticides capability to induce oxidative stress in humans and animals has been profuse. Considering that antx-a(s) is the unique organophosphate produced by cyanobacteria currently known; the main of this work was to investigate the biochemical mechanism related to the pro-oxidant capacity of antx-a(s)-containing extracts. In order to achieve the objective, there was determined the activity of cholinesterasic and antioxidant enzymes such as acetylcholinesterase (AChE), butirylcholinesterase (BuChE), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD). Two experimental models were used: (i) Swiss mice were treated intraperitoneally (ip) with 20 mg.kg-1 of antx-a(s)-containing extract and sacrificed after 24 h, 48 h, 7 and 14 days of treatment and, (ii) alfalfa seeds were irrigated with antx-a(s)-containing extract for 7 days. Additionally, the cardiac effect of antx-a(s)-containing extract on Leurolestes circunvagans cockroach was evaluated. The AChE and BuChE activity in mice treated with antx-a(s)-containing extract stayed inhibited more than 55% during 48 h (n=9, p<0,001). Normal activity of both enzymes was observed after seven days of treatment. By the other hand, changes in the antioxidant enzymes activity only began after the second day of treatment. Initially, both CAT and GPx showed lower activity than the control group after 48 h. Among those enzymes, GPx showed the highest decreased activity (n=9, p<0,001). After seven days, while the antx-a(s)- containing...

Effect of Anatoxin-a on Expression of IEGs Genes in Mouse Brain / 环境与健康杂志

Objective To explore the role of IEGs in the central neurotoxicity of mice induced by anatoxin-a ANTX-A. Methods 80 Kunming mice were divided into groups and treated with ANTX-A at 200, 50 and 12.5 ?g/kg respectively i.p. for one month, 0.9% sodium chloride solution was used as the control. The expression of c-fos and NGFI-A genes and CREB protein in mouse brain were determined by using RT-PCR and immunocytochemical methods. Results The expression of c-fos gene significantly increased in the females of 50, 200 ?g/kg groups and in the males of 200 ?g/kg group compared with the control group. The expression of C-FOS and CREB-1 protein was significantly higher in 200 ?g/kg group than that in the control group. In all ANTX-A treated groups, the expression of NGFI-A gene did not show a significant difference compared with the control. Conclusion c-fos and CREB-1 genes may play a role in the central neurotoxicity induced by anatoxin-a.

Effect of Anatoxin-a on the Activity of ATPase in PC12 Cells / 环境与健康杂志

Objective To study the effects of anatoxin-a (ANTX-A) on the activity of ATPase in PC12 cells.Methods PC12 cells were activated by different concentrations of ANTX-A stimulation for an hour or 10-7 mol/L ANTX-A for different time.PC12 cells were desensitized using twice incubation method.The activities of Na+-K+-ATPase and Ca2+-ATPase were determined by colorimetry method.Results 10-9,10-8,10-7 mol/L ANTX-A activated PC12 cells for an hour and 10-7 mol/L ANTX-A activated PC12 cells for 30,60,120 min,the activity of Na+-K+-ATPase in PC12 cells,in all of the cases,showed an reduction tendency,the activity of Ca2+-ATPase decreased significantly in different activation situation compared to the control group(P