The use of histológical parameters to assess intestinal and liver health on broilers challenged isolatedly and simultaneously with cyclopiazonic acid and aflatoxin / B1 El uso de parámetros histológicos para evaluar la salud intestinal y hepática en pollos de engorde desafiados de forma aislada y simultánea con ácido ciclopiazónico y aflatoxina B1

Abstract Mycotoxins contaminate agricultural commodities, which contaminates animals. These toxins can damage vital organs, such as the liver, as well as the epithelial tissue. Among these mycotoxins are aflatoxin B1 (AFB1) and cyclopiazonic acid (CPA), which can occur simultaneously in food. In broilers, mycotoxicosis has an economic impact due to several factors, such as low feed conversion rate, incidence of other diseases, and interference with reproductive capacity, all of which may lead to a public health problem. The aim of the present study was to histologically assess, through the I See Inside (ISI) method, harmful effects on broiler liver, duodenum, jejunum, and ileum in the presence of AFB1 and CPA isolatedly and simultaneously. Groups challenged with mycotoxins showed significant damage to both gut and liver fragments. All challenged-groups in all fragments impaired the parameters analyzed for intestinal epithelium. In the liver, AFB1 was predominantly harmful when the parameters were analyzed separately, but when analyzing the total ISI score, CPA was also found to be harmful to this organ. The other point analyzed was the great variation between the weights of the birds contaminated by mycotoxin while the negative control group presents a lesser variation.

Resumen Las micotoxinas contaminan los productos agrícolas, que a su vez contaminan a los animales. Estas toxinas pueden dañar órganos vitales, como el hígado y el tejido epitelial. Entre estas micotoxinas se encuentran la aflatoxina B1 (AFB1) y el ácido ciclopiazónico (CPA), que pueden hallarse simultáneamente en los alimentos. En los pollos de engorde, la micotoxicosis tiene un impacto económico debido a varios factores, como la baja tasa de conversión alimenticia, la incidencia de otras enfermedades y la interferencia de la capacidad reproductiva, que pueden llevar a un problema de salud pública. El objetivo de la presente investigación es la de evaluar histológicamente, a través del método "I See Inside" (ISI), los efectos nocivos sobre el hígado, duodeno, yeyuno e íleon de pollos de engorde en presencia de AFB1 y CPA de forma aislada y simultánea. Los grupos desafiados con micotoxinas presentaron un daño significativo tanto en el intestino como en los fragmentos del hígado. Todos los grupos tratados tuvieron alteraciones en los parámetros analizados para el epitelio intestinal. En el hígado, AFB1 fue predominantemente dañino cuando los parámetros se analizaron por separado, pero al examinar la puntuación ISI total, también se encontró que el CPA era perjudicial para este órgano. Otra cuestión que fue investigada fue la gran variación entre los pesos de las aves contaminadas por micotoxinas mientras el grupo de control negativo presentó una variación menor.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach / Identificação de espécies toxigênicas de Aspergillus isoladas da dieta de cabras leiteiras utilizando uma abordagem polifásica

Some species of filamentous fungi that infest agricultural commodities are able to produce mycotoxins, contaminating feed and animal products. The aim of this research was to identify the mycoflora present in the feed and forage for dairy goat and to isolate and characterize the Aspergillus flavus and A. parasiticus strains based on a morphological and molecular characterization and mycotoxigenic ability. The goat dairy diets were collected monthly from 11 goat milk farms, totaling 129 and 106 samples of concentrate and forage, respectively. For the isolation of the mycobiota the surface plating method was used. Aspergillus, Penicillium, and Fusarium were the main fungi producing mycotoxins isolated. The morphological and molecular characterization and mycotoxigenic ability were used for A. flavus and A. parasiticus identification. The Aspergillus spp. from feed 39% produced aflatoxins B1 and B2, 17% produced cyclopiazonic acid (CPA), 18% produced both toxins, and 42% had no toxigenic ability. Only 2.0% of the strains produced aflatoxins B1, B2, G1, and G2, but no CPA. The strains from forage were producers of aflatoxins B1 and B2 (37%), CPA (14%), 14% of both mycotoxins, whereas 49% have shown no toxigenic ability. The aflD and aflR genes were used by PCR and PCR-RFLP, respectively. The presence of toxigenic species in samples of feed for lactating goats indicates a potential risk of contamination of dairy products, if they are exposed to environmental conditions favorable to fungal growth and mycotoxin production.

Algumas espécies de fungos filamentosos que infestam os produtos agrícolas e ração são capazes de produzir micotoxinas. O objetivo deste trabalho foi identificar a micoflora presente nos concentrados e volumosos utilizados na dieta de cabras leiteiras e isolar as espécies Aspergillus flavus e A. parasiticus, com base em uma caracterização morfológica e molecular e capacidade micotoxigênica. Os alimentos foram coletados mensalmente em 11 fazendas produtoras de leite de cabra, totalizando 129 e 106 amostras de concentrado e volumoso, respectivamente. Para o isolamento da micobiota, foi utilizado o método de plaqueamento de superfície. Aspergillus, Penicillium e Fusarium foram os principais gêneros de fungos produtores de micotoxinas isolados das amostras. A caracterização morfológica e molecular e capacidade micotoxigênica foram utilizadas para identificação de A. flavus e A. parasiticus. Das cepas Aspergillus spp isoladas do concentrado, 39% produziram aflatoxinas B1 e B2, 17% produziram ácido ciclopiazônico (ACP), 18% produziram ambas as toxinas e 42% não tinham capacidade toxigênica. Apenas 2,0% das cepas produziram aflatoxinas B1, B2, G1 e G2. As cepas de Aspergillus spp. isoladas do volumoso foram produtores de aflatoxinas B1 e B2 (37%), ACP (14%), sendo que 14% produziram ambas toxinas e 49% não foram produtoras. Os genes aflD e aflR foram utilizados para a PCR e a PCR-RFLP, respectivamente. A presença de espécies toxigênicas em amostras de alimentos destinados a caprinos em lactação indica um risco potencial de contaminação dos produtos lácteos por aflatoxinas e ACP, caso estes sejam expostos a condições ambientais favoráveis ao crescimento de fungos e produção de micotoxinas.

Effect of SKF96365 and NiCl2 on cyclopiazonic acid induced intracellular calcium cation concentration increase in rat distal pulmonary arterial smooth muscle cells / 重庆医学

Objective To study the effect of SKF96365 and NiCl2 on cyclopiazonic acid (CPA) induced intracellular calcium cation concentration ([Ca2+ ]i ) change in rat distal pulmonary arterial smooth muscle cells (PASMC) .Methods The rat distal PASMC were isolated and cultured .The effects of CPA ,SKF96365 and NiCl2 on [Ca2+ ]i in PASMC were tested by fluorescence microscope and InCyte [Ca2+ ]i measurement system .Results PASMC were incubated with Ca2+‐free Krebs solution containing 5μmol/L nifedipine ,10 μmol/L CPA caused a small transient increase in [Ca2+ ]i ;after restoration of extracellular Ca2+ to 2 .5 mmol/L ,10 μmol/L CPA caused marked increases in [Ca2+ ]i in PASMC incubated with Krebs solution containing 5 μmol/L nife‐dipine .Both 50 μmol/L SKF96365 and 500 μmol/L NiCl2 distinctly attenuated the increases in [Ca2+ ]i caused by 10 μmol/L CPA in PASMC .However ,neither 50 μmol/L SKF96365 nor 500 μmol/L NiCl2 affected the increases in [Ca2+ ]i caused by 60 mmol/L KCl in PASMC .Conclusion CPA induced increases in [Ca2+ ]i may related to Ca2+ release from sarcoplasmic reticulum and the in‐flux of Ca2+ through store‐operated Ca2+ channels (SOCC) in rat distal PASMC .Both SKF96365 and NiCl2 could selectively block SOCC and attenuated the influx of Ca2+ through SOCC in PASMC .

Effect of Acute Hypoxia on Cyclopiazonic Acid Induced Intracellular Calcium Cation Enhancement in Rat Distal Pulmonary Venous Smooth Muscle Cells / 中国循环杂志

Objective: To study the effect and the mechanism of acute hypoxia on Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA) induced intracellular calcium cation enhancement in rat distal pulmonary venous smooth muscle cells (PVSMC) . Methods: The PVSMC were isolated from 6 male SD rats and the cells were cultured for further experiment. Enhancing effects of CPA, acute hypoxia (4% O2) on [Ca2+]i in distal PVSMC and the interventional effects of 2 store-operated Ca2+ channels (SOCC) inhibitors, NiCl2 and SKF96365 on [Ca2+]i in distal PVSMC were tested by lfuorescence microscope and intracellular [Ca2+] examining system. Results: When PVSMC were perfused with Ca2+-free Krebs solution containing 5 μmol/L nifedipine, 10 μmol/L CPA caused a slight elevation of [Ca2+]i, and acute hypoxia obviously enhanced the [Ca2+]i in PVSMC. When restoration of extracellular [Ca2+] to 2.5 mmol/L, 10 μmol/L CPA caused signiifcant elevation of [Ca2+]i, and acute hypoxia obviously enhanced [Ca2+]i induced by CPA in PVSMC. The SOCC inhibitors, NiCl2 (500 μmol/L) and SKF96365 (50 μmol/L) distinctively attenuated the elevation of [Ca2+]i by hypoxia and CPA. However, NiCl2 and SKF96365 had no effect on high potassium (60 mmol/L KCl Krebs solution) induced elevation of [Ca2+]i in distal PVSMC. Conclusion: Acute hypoxia enhanced the elevation of [Ca2+]i induced by CPA; such effect could be selectively blocked by SOCC inhibitor which indicated that acute hypoxia could enhance the activity of SOCC in rat distal PVSMC.

Avaliação da micoflora e ocorrência de micotoxinas em cascas de amendoim em diferentes estágios de maturação da vagem / Evaluation of mycoflora and occurrence of mycotoxins in peanut hulls at different pod maturation stages

As cascas de amendoim (Arachis hypogaea L.) são de grande importância para confecção de cama de frangos, de gado de leite e como fonte de fibras para ruminantes, portanto a elucidação dos mecanismos de contaminação por fungos toxigênicos e por micotoxinas em amendoim é imprescindível, especialmente para que medidas preventivas possam ser tomadas. Realizou-se, este trabalho, em Junqueirópolis, Estado de São Paulo, Brasil. Os principais fungos isolados nas cascas de amendoim foram Fusarium ssp. (78,75 por cento), Rhizopus ssp. (14,1 por cento) e A. flavus (11,75 por cento). No solo foram isolados Penicillium spp., Fusarium spp. e Aspergillus flavus, entre outros. Aflatoxinas foram detectadas em amostras de cascas de amendoim a partir do estágio de granação em concentrações que variaram de 5,42 μg/kg a 218,52 μg/kg. Ácido ciclopiazônico e fumonisinas B1 e B2 não foram detectadas. A presença de A. flavus e aflatoxinas nas amostras, revela a importância de um controle das cascas de amendoim antes de sua utilização. Boas práticas agrícolas são indicadas para região, uma vez que a contaminação das vagens ocorreu antes da colheita.

Peanut (Arachis hypogaea L.) hulls are very important because they are used as litter to poultry and dairy cattle and as fiber source to cattle. The elucidation of the peanuts contamination mechanisms by toxigenic fungi and their mycotoxins is vital, specially for prevention measurements. The peanuts total mycoflora and mycotoxin contamination were analyzed in plants sampled in Junqueirópolis, in São Paulo State (Brazil) at different stages of the pod maturity. The prevalent mycoflora in peanut hulls were Fusarium spp., Rhizopus spp. and Aspergillus flavus. In soil under the peanut crop, the genus Penicillium spp., Fusarium spp. and A. flavus were detected. Aflatoxins were detected in peanut hull samples since filling pod stage in concentrations from 5.42 μg/kg to 218.52 μg/kg. Cyclopiazonic acid and fumonisins were not detected. The A. flavus presence and the detection of aflatoxins indicate the importance of quality control of peanut hulls before their utilization, and the adoption of agricultural practices showed to avoid the contamination since the peanut pods contamination happened before the harvest.

Regulation of Ba2+-Induced Contraction of Murine Ureteral Smooth Muscle

This study was designed to characterize ureteral smooth muscle motility and also to study the effect of forskolin (FSK) and isoproterenol (ISO) on smooth muscle contractility in murine ureter. High K+ (50 mM) produced tonic contraction by 0.17+/-0.06 mN (n=19). Neuropeptide and neurotransmitters such as serotonin (5microM), histamine (20microM), and carbarchol (CCh, 10~50microM) did not produce significant contraction. However, CCh (50microM) produced slow phasic contraction in the presence of 25 mM K+. Cyclopiazonic acid (CPA, 10microM), SR Ca2+-ATPase blocker, produced tonic contraction (0.07 mN). Meanwhile, inhibition of mitochondria by protonophore carbnylcyanide m-chlorophenylhydrazone (CCCP) also produced weak tonic contraction (0.01 mN). The possible involvement of K+ channels was also pursued. Tetraethyl ammonium chloride (TEA, 10 mM), glibenclamide (10microM) and quinidine (20 microM) which are known to block Ca2+-activated K+ channels (KCa channel), ATP-sensitive K+ channels (KATP) and nonselective K+ channel, respectively, did not elicit any significant effect. However, Ba2+ (1~2 mM), blocker of inward rectifier K+ channels (KIR channel), produced phasic contraction in a reversible manner, which was blocked by 1microM nicardipine, a blocker of dehydropyridine-sensitive voltage-dependent L-type Ca2+ channels (VDCCL) in smooth muscle membrane. This Ba2+-induced phasic contraction was significantly enhanced by 10microM cyclopiazonic acid (CPA) in the frequency and amplitude. Finally, regulation of Ba2+-induced contraction was studied by FSK and ISO which are known as adenylyl cyclase activator and beta-adrenergic receptor agonist, respectively. These drugs significantly suppressed the frequency and amplitude of Ba2+-induced contraction (p<0.05). These results suggest that Ba2+ produces phasic contraction in murine ureteral smooth muscle which can be regulated by FSK and beta-adrenergic stimulation.

Effects of S-nitrosocaptopril on cyclopiazonic acid-induced [Ca~(2+) ]_i change in rat aortic smooth muscle cells / 中国药理学通报

AIM To study the characteristics of Ca 2+ channel mediated store operated Ca 2+ influx on rat vascular smooth muscle. METHOD Fura 2 fluorescence technique was used to investigate the [Ca 2+ ] i change. RESULTS ① S nitrosocaptopril (CapNO,20～120 ?mol?L -1 ) produced a concentration dependent inhibitory effect on cyclopiazonic acid(CPA) induced [Ca 2+ ] i change. The maximal inhibitory effect(37%?17%) of CapNO was reached at a concentration of 80 ?mol?L -1 . The same concentration of Captopril had no effects. ② Inhibition rate of 80 ?mol?L -1 CapNO (The concentration of maximal effect, CME) on CPA induced [Ca 2+ ] i change was 30%?10%, subsequent addition of 1 ?mol?L -1 Nif (CME)did not further produced the decrease effect (54%?18%). subsequent addition of 20 ?mol?L -1 SK&F96365 (CME) further produced the decrease effect. The inhibitory effects of 20 ?mol?L -1 SK&F96365 were significantly different in the cases of CapNO and Nif pretreatment(24%?10%) and non treatment (54%?11%). ③ The inhibitory effects of 2 ?mol?L -1 tyrphostinAG490(AG490,CME) were significantly different in the cases of CapNO (CME) pretreatment (24%?9%)and non treatment (42%?10%). 80 ?mol?L -1 CapNO effect on CPA induced [Ca 2+ ] i changes in AG490 pretreatment condition(18%?7%) was different from that in non treatment case(37%?10%). CONCLUSION S nitrosocaptopril obviously inhibits the opening of SOCC and VDCC, which mediates store operated Ca 2+ influx. The inhibitory effects of CapNO is associated with both sensitive to and non sensitive to tyrosine kinase (Janus2).

Cyclopiazonic acid enhanced the Ca~(2+)-dependent outward K~+ currents in cultural aortic smooth muscle cells from SHR and WKY / 中国药理学通报

ABSTRACT The effects of cyclopiazonic acid (CPA) on the Ca2+ -dependent ourward K+-cur-rent [IK(ca)] were studied using whole-cell and single-channel patch-clamp techniques. Depolarization (pipette potential range from - 20 to - 120 mV)induced a outward Ik(ca) with a conductance of about 40 pS in the cultural aortic smooth muscle cells from SHR and WKY. 10 ?mol ? L-1 CPA significantly enhanced these currents and prolonged the mean open time of the channels. This effect of CPA was completely blocked by glybenclamide, a K+-channel block-er. In the whole cell recording experiments, CPA increased the amplitude of outward K+-current. This effect of CPA was Ca2+-dependent and completely blocked by glybenclamide. There was no any significant difference between the effects of CPA in SHR and in WKY. These results suggest that the functional change of vascular smooth muscle in SHR doesn't appear to be related to Ik(ca).