Análise físico-química e atividades antifúngica e antioxidante do óleo essencial da flor de Curatella americana L / Physichochemical analysis, and antifungal and antioxidant activities of the essential oil of Curatella americana L. flower

Introdução: a espécie vegetal Curatella americana produz anualmente inflorescências com aroma adocicado rica em óleo essencial. Objetivo: avaliar as características físico-químicas, e atividades antifúngica e antioxidante do óleo essencial da flor de Curatella americana. Metodologia: as flores foram coletadas em quatro áreas de Cerrado no estado de Goiás; o rendimento de óleo essencial foi obtido através de hidrodestilação; as características físicas foram determinadas para densidade e solubilidade, a atividade antioxidante foi determinada pela redução do radical livre DPPH; a atividade antifúngica foi determinada por inibição das cepas de Candida, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides e Aspergillus flavus. Resultados: o rendimento de óleo foi de 0,18%, densidade de 0,907 g mL-1, solubilidade positiva para EtOH 70%, atividade antioxidante de CI50 µL mL-1 1,95. Atividade de inibição fúngica apenas para Candida tropicalis na concentração de 8% com halo de antibiose de 10 mm. Sensibilidade discreta nas maiores concentrações de 25, 50 e 100 µL-1 para Aspergillus flavus e Sclerotinia sclerotiorum e baixa atividade de inibição para Colletotrichum gloeosporioides. Conclusão: o óleo essencial da flor de Curatella americana apresentou baixo rendimento, entretanto, alta eficiência na redução do radical livre DPPH. As atividades antifúngicas apresentaram bons resultados de inibição, entretanto, torna-se necessário a adição de outros óleos essenciais para aumento das taxas de inibição micelial.

Introduction: the plant species Curatella americana produces annual inflorescences with a sweet flavour rich in essential oil. Objective: to evaluate the physicochemical characteristics, antifungal and antioxidant activities of the essential oil of the Curatella americana flower. Methodology: the flowers were collected in four areas of Cerrado in the state of Goiás; the essential oil yield was obtained through hydrodistillation; the physical characteristics were determined for density and solubility, the antioxidant activity was determined by the reduction of the free radical DPPH; antifungal activity was determined by inhibiting the strains of Candida, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides and Aspergillus flavus. Results: the oil yield was 0.18%, density 0.907 g mL-1, positive solubility for EtOH 70%, antioxidant activity of IC50 µL mL-1 1.95. Fungal inhibition activity only for Candida tropicalis at a concentration of 8% with a 10 mm antibiosis halo. Discrete sensitivity in the highest concentrations of 25, 50 and 100 µL-1 for Aspergillus flavus and Sclerotinia sclerotiorum and low inhibition activity for Colletotrichum gloeosporioides. Conclusion: The essential oil of the Curatella americana flower showed low yield, however, high efficiency in reducing DPPH free radical. Antifungal activities showed good inhibition results, however, it is necessary to add other essential oils to increase mycelial inhibition rates.

Aflatoxin levels and prevalence of TP53 aflatoxin-mutations in hepatocellular carcinomas in Mexico / Niveles de aflatoxina y prevalencia de mutaciones del gen TP53 por aflatoxina en carcinoma hepatocelular en México

Abstract: Objective: To determine the exposure to aflatoxin B1 (AFB1) in southern Mexico and the presence of the aflatoxin signature mutation in hepatocellular carcinoma (HCC) tissue from patients from a cancer referral center. Materials and methods: We estimated the prevalence and distribution of AFB1 in a representative sample of 100 women and men from Chiapas using the National Health and Nutrition Survey 2018-19. We also examined the presence of the aflatoxin signature mutation in codon 249 (R249S), and other relevant mutations of the TP53 gene in HCC tissue blocks from 24 women and 26 men treated in a national cancer referral center. Results: The prevalence of AFB1 in serum samples was 85.5% (95%CI 72.1-93.1) and the median AFB1 was 0.117 pg/µL (IQR, 0.050-0.350). We detected TP53 R249S in three of the 50 HCCs (6.0%) and observed four other G>T transversions potentially induced by AFB1. Conclusion: Our analysis provides evidence that AFB1 may have a relevant role on HCC etiology in Mexico.

Resumen: Objetivo: Determinar la exposición a aflatoxina_B1 (AFB1) en el sur de México y la presencia de la mutación característica de AFB1 en tejido de carcinoma hepatocelular (CHC) de pacientes de un centro oncológico. Material y métodos: Se estimó la prevalencia y distribución de AFB1 en una muestra representativa de 100 mujeres y hombres de Chiapas a partir de la Encuesta Nacional de Salud y Nutrición 2018-19. También se observó la presencia de la mutación característica de AFB1 en el codón 249 (R249S), y otras mutaciones relevantes del gen TP53 en bloques de tejido de CHC de 24 mujeres y 26 hombres estudiados en un centro de referencia nacional de oncología. Resultados: La prevalencia de AFB1 en las muestras de suero fue de 85.5% (IC95% 72.1-93.1) y la mediana de la concentración 0.117 pg/µL (IQR, 0.050-0.350). Se detectó TP53 R249S en tres de 50 casos de CHC (6.0%) y se observaron cuatro transversiones G>T potencialmente inducidas por AFB1. Conclusión: El presente análisis proporciona evidencia de que la AFB1 puede tener un papel relevante en la etiología del CHC en México.

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.

Detección de aflatoxina M1 en muestras de leche cruda de vacas en tanques de enfriamiento en Boyacá (Colombia) / Detection of aflatoxin M1 in raw cow milk samples in cooling tanks in Boyacá (Colombia)

RESUMEN Una de las principales formas de contaminación de la leche con micotoxinas es el consumo de alimentos fermentados que se encuentran contaminados con mohos principalmente de Aspergillus spp., los cuales producen toxinas que pueden llegar a constituirse como un problema para la salud publica debido a su estabilidad térmica y química. El objetivo del presente trabajo fue detectar las concentraciones de aflatoxina M1 en muestras de leche de vacas en tanques de enfriamiento en cuatro municipios del departamento de Boyacá durante un año, determinando las variaciones de acuerdo con la temporada. Se realizó un estudio de corte longitudinal, descriptivo cuantitativo. Se seleccionaron aleatoriamente cuatro tanques de enfriamiento de cuatro municipios distintos del departamento; cada uno se muestreó dos veces al mes durante todo el período de estudio y se procesaron mediante metodología Charm Ez Lite . Se realizó un ANDEVA para determinar las diferencias estadísticas entre las concentraciones de la aflatoxina M1 por cada trimestre. Se determinaron diferencias estadísticas entre cada uno de los trimestres del estudio encontrando un porcentaje de positividad de 74,06% del total de muestras positivas en los trimestres de verano. 28,12% (108) de las muestras tomadas durante todo el estudio fueron positivas, con concentraciones de la toxina que oscilaron entre 0,5 y 2,0 μg/Kg de leche. Se determinó por primera vez en el departamento de Boyacá las concentraciones y variaciones estacionales de aflatoxina M1 en muestras de tanques de enfriamiento de leche, encontrando las mayores concentraciones y número de casos positivos de aflatoxina M1 en los meses de verano.

ABSTRACT One of the main forms of contamination of milk with mycotoxins is the consumption of fermented foods that are contaminated with mold, mainly Aspergillus spp, which produce toxins that can become a public health problem due to their thermal and chemical stability. The objective of the present work was to detect aflatoxin M1 concentrations in cows' milk samples in cooling tanks in four municipalities of the department of Boyacá for one year, determining the variations according to the season. A longitudinal, quantitative descriptive study was carried out, four cooling tanks from four different municipalities in the department were randomly selected, each tank, in each municipality, was sampled twice a month throughout the study period and processed using Charm methodology Ez Lite®, an ANDEVA was performed to determine the statistical differences between aflatoxin M1 concentrations for each quarter. Statistical differences were determined between each of the quarters of the study, finding a positivity percentage of 74.06% of the total positive samples in the summer quarters. 28.12% (108) of the samples taken throughout the study were positive, with toxin concentrations ranging between 0.5 and 2.0 μg/Kg of milk. Seasonal concentrations and variations of aflatoxin M1 in milk cooling tank samples were determined for the first time in the department of Boyacá, finding the highest concentrations and number of positive cases of aflatoxin M1 in the summer months.

Biopanning the mimotopes of aflatoxin B1 and their immunogenicity / Panning de mimotopo aflatoxina B1 e sua imunogenicidade

Utilizando um anticorpo monoclonal contra a aflatoxina B1 (AFB1) como ligante, foi identificado um mimotopo específico de aflatoxina B1 após se realizarem quatro ciclos de seleção biológica de 7-peptídeos aleatórios em biblioteca de fago exibida. O mimotopo é denominado P10, e sua sequência de aminoácidos é YRRHEKD. O soro imunológico de ratos Balb/c imunizados com P10 foi especificamente ligado à aflatoxina B1-albumina, indicando que o anticorpo era específico ao AFB1. Esses resultados sugerem que é possível desenvolver a vacina baseada em mimotopo associado à toxina.(AU)

El factor de transcripción bZIP Afap1 afecta al estrés oxidativo y la biosíntesis de aflatoxinas en Aspergillus flavus / The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus

Abstract Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap 1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H 2O 2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H 2O 2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB 1 in the mutant strains was approximately V of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus. ©2018 Published by Elsevier España, S.L.U. on behalf of Asociación Argentina de Microbiología. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

Resumen La aflatoxina es un metabolito secundario cancerígeno producido principalmente por Aspergillus flavus y Aspergillus parasiticus, que pone en riesgo grave a la salud de los humanos y los animales. El estrés oxidativo es una respuesta de defensa común, y es sabido que las especies reactivas de oxígeno (ROS) pueden inducir la síntesis de una serie de metabolitos secundarios, incluida la aflatoxina. Empleando mutantes carentes del gen afap1 se evaluó el papel de Afap1 en el estrés oxidativo y la síntesis de aflatoxinas. El crecimiento de las cepas mutadas se vio significativamente inhibido con el aumento de la concentración de H 2O 2, la inhibición fue completa a 40mmol/l. Sin embargo, en el análisis cuantitativo por HPLC, la concentración de la aflatoxina AFBi aumentó con el aumento de la concentración de H 2O 2 hasta 10mmol/l. Tras un análisis apoyado en la información provista por la herramienta NCBI BLAST, se supuso que Afap1, un factor de transcripción de la cremallera de leucina básica (bZIP), estaba asociado con el estrés oxidativo en este hongo. El tratamiento con 5mmol/l de H 2O 2 inhibió completamente el crecimiento de las cepas mutantes en afap1, pero no afectó el crecimiento de la cepa CA14PTs (cepa no mutada). Además, la concentración de AFB 1 en las cepas mutadas fue de aproximadamente 1/4 de la observada en CA14PTs. Estos resultados sugieren que Afap1 juega un papel clave en la regulación del estrés oxidativo y la producción de aflatoxinas en A. flavus.

Mutación R249S TP53 en pacientes con cirrosis y carcinoma hepatocelular en un hospital de Medellín / TP53 R249S mutation in patients with cirrhosis and hepatocellular carcinoma in a hospital at Medellin

Resumen Introducción: la exposición dietaria a la aflatoxina es un factor de riesgo para carcinoma hepatocelular, el cáncer primario de hígado más frecuente. Esta asociación se estableció gracias a la evidencia in vitro e in vivo de la relación entre la exposición a la aflatoxina B1 y la transversión G→T en el codón 249 del gen TP53, así como evidencia de la sinergia entre la aflatoxina y la infección crónica por virus de la hepatitis B. Métodos: se determinó la frecuencia de la mutación R249S del gen TP53 en 30 pacientes con diagnóstico de cirrosis y/o carcinoma hepatocelular quienes fueron sometidos a trasplante hepático en un hospital en Medellín, Colombia. Se extrajo ADN a partir de las muestras de explante hepático, se amplificó el fragmento de interés y se detectó la mutación por polimorfismos de longitud de fragmentos de restricción. Resultados: se encontró la mutación R249S en una de las 30 muestras analizadas (3,33 %) y se determinó, por medio de marcadores serológicos, infección por el virus de la hepatitis B en dos casos (6,67 %). No se encontró simultáneamente la mutación y la presencia de los marcadores de infección por virus de la hepatitis B. Conclusión: los resultados sugieren una baja exposición dietaria con aflatoxina B1 en la población de estudio. Sin embargo, es importante tener en cuenta la regulación de los límites permisibles de aflatoxina B1 y la inclusión en el diagnóstico diferencial de carcinoma hepatocelular, dada la heterogeneidad de las condiciones de la población en diferentes regiones del país.

Abstract Introduction: The dietary exposure to aflatoxin is a risk factor of hepatocellular carcinoma, the most frequent primary liver cancer. This risk factor was identified after in vivo and in vitro evidence of the relation between exposure to aflatoxin B1 and transversion G → T at 249 codon of the TP53 gene; as well as evidence of the synergy between hepatitis B virus chronic infection. Methods: the frequency of the R249S mutation of the TP53 gene was determined in 30 cases of cirrhosis and/or hepatocellular carcinoma, with liver transplantation in the hepatology unit of a hospital in Medellín, Colombia. DNA was extracted from the liver explant samples; the sequence of interest was amplified, and the mutation was detected by restriction fragment length polymorphisms. Results: the R249S mutation was found in 1 of the 30 samples analyzed (3.33 %); and hepatitis B virus infection was detected by serological markers in 2 of the 30 cases (6.67 %). We did not find the mutation and the presence of hepatitis B virus infection markers at the same time in any of the samples. Conclusion: The results suggest a low dietary exposure with aflatoxin B1 in the study population. However, it is important to take into consideration the regulation of the permissible limits of aflatoxin B1 and the inclusion in the differential diagnosis of hepatocellular carcinoma, given the heterogeneity of the conditions of the population in different regions of the country.

Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver / Validación de una técnica para detectar aflatoxina B1 en hígado de pollo por cromatografía líquida de alta performance acoplada a espectrómetro de masas en tándem

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus. It is the predominant mycotoxin found in raw materials used for the manufacture of broiler feeds. The aim of the present study was to develop a new and optimized method for the detection and quantification of aflatoxin B1 (AFB1) residues in broiler liver using solid phase extraction (SPE) clean-up and liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) detection. The method was validated for linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The validation parameters indicated satisfactory linearity (r² >0.99), accuracy and precision (4.57% intra-day RSD; 14.65% inter-day RSD) a very high recovery (99 ±13%) and high sensitivity achieved for AFB1 in animal samples (LOD = 0.017 and LOQ= 0.050 ng/g). The method was effective for the detection and quantification of AFB1 residues in broiler liver and could also be potentially used for detecting AFB1 in other edible animal tissues after natural or experimental AFB1 exposure with high sensitivity and precision.

La aflatoxina B1 (AFB1) es una micotoxina carcinogénica y mutagénica producida principalmente por Aspergillus flavus y Aspergillus parasiticus. Es la principal toxina que contamina las materias primas utilizadas para la elaboración de alimentos balanceados destinados a la alimentación de pollos parrilleros. El objetivo de este trabajo fue desarrollar un método nuevo y optimizado para detectar y cuantificar bajos niveles de AFB1 en hígado de pollo, usando limpieza por extracción en fase sólida (SPE) y cromatografía líquida acoplada a detección por espectrometría de masa en tándem con ionización por electrospray (LC-ESI-MS/MS). Se validaron la linealidad, la exactitud, la precisión, el límite de detección (LOD) y el límite de cuantificación (LOQ). El método resultó tener linealidad (r²>0,99), exactitud y precisión muy satisfactorias (4,57% RSD intradía; 14,65% RSD interdía), un alto porcentaje de recupero (99 ± 13%) y la sensibilidad más alta lograda para la detección de AFB1 en muestras de origen animal (LOQ=0.050 ng/g y LOD = 0.017). El método fue muy efectivo para detectar y cuantificar bajos niveles de AFB1 en hígados de pollos parrilleros. Este método podría potencialmente utilizarse para la detección de esta toxina en otros tejidos y subproductos de origen animal luego de su exposición a AFB1 con una mayor sensibilidad y precisión.

Efectos de la temperatura, la actividad de agua y el tiempo de incubación en el crecimiento fúngico y la producción de aflatoxina B1 por aislados toxicogénicos de Aspergillus flavus en sorgo / Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds

Sorghum, which is consumed in Tunisia as human food, suffers from severe colonization by several toxigenic fungi and contamination by mycotoxins. The Tunisian climate is characterized by high temperature and humidity that stimulates mold proliferation and mycotoxin accumulation in foodstuffs. This study investigated the effects of temperature (15, 25 and 37 °C), water activity (a w, between 0.85 and 0.99) and incubation time (7, 14, 21 and 28 d) on fungal growth and aflatoxin B1 (AFB1) production by three Aspergillus flavus isolates (8, 10 and 14) inoculated on sorghum grains. The Baranyi model was applied to identify the limits of growth and mycotoxin production. Maximum diameter growth rates were observed at 0.99 a w at 37 °C for two of the isolates. The minimum a w needed for mycelial growth was 0.91 at 25 and 37 °C. At 15 °C, only isolate 8 grew at 0.99 a w. Aflatoxin B1 accumulation could be avoided by storing sorghum at low water activity levels (≤0.91 a w). Aflatoxin production was not observed at 15 °C. This is the first work on the effects of water activity and temperature on A. flavus growth and AFB1 production by A. flavus isolates on sorghum grains.

El sorgo, que se consume en Túnez como alimento humano, puede sufrir la colonización severa de varios hongos toxicogénicos, con la consiguiente bioacumulación de micotoxinas. Además, el clima de Túnez, caracterizado por las altas temperaturas y humedad, estimula el crecimiento fúngico y la acumulación de micotoxinas en los productos alimenticios. Este estudio investigó los efectos de la temperatura (15, 25 y 37 °C), la actividad de agua (a w) (entre 0,85 y 0,99) y el tiempo de incubación (7, 14, 21 y 28 días) sobre el crecimiento y la producción de aflatoxina B1 (AFB1) de 3 aislados de Aspergillus flavus (designados como 8, 10 y 14) que se inocularon sobre granos de sorgo. El modelo Baranyi se aplicó para identificar los límites del crecimiento y la producción de micotoxinas. Las tasas máximas de crecimiento para 2 de los aislados se observaron en la combinación 0,99 a w y 37 °C. La a w mínima necesaria para el crecimiento del micelio fue de 0,91 a 25 °C y 37 °C. A 15 °C, solo el aislado 8 creció a 0,99 a w, pero fue incapaz de producir la aflatoxina B1. Es posible evitar la acumulación de aflatoxina B1 en el sorgo almacenándolo a baja actividad de agua (≤ 0,91 a w). Este es el primer trabajo que ha estudiado el efecto de la actividad del agua y la temperatura sobre el crecimiento de aislados de A. flavus y su producción de aflatoxina B1 en granos de sorgo.

Aflatoxin M1 in pasteurized, UHT milk and milk powder commercialized in Londrina, Brazil and estimation of exposure / Aflatoxina M1 em leite pasteurizado e UHT e leite em po comercializados em Londrina, Brasil e exposicao estimada.

Aflatoxin M1 (AFM1) is found in milk and other excretion products after aflatoxin B1 intake. AFM1 is carcinogenic to humans, and known levels of dairy product contamination is important to understand the risks to which the population is exposed. The occurrence of AFM1 was evaluated in 42 milk samples commercialized in Londrina, Parana State, Brazil and this rate of occurrence was used to estimate this exposure. AFM1 determina tion was ca rried out by ELISA, and was detected in 100 % samples at levels ranging from 0.01 to 0.81 µ g/kg (mean 0.13 µ g/kg). None of the samples p resente d AFM1 above the maximum permitted level by Brazilian Legislation (0.5 µ g/kg for fluid milk and 5 µ g/kg for milk powder). The estimated daily intake (EDI) of AFM1 was evaluated, and the average intake was 0.468 ng/kg body weight (b.w.) for adolescents, 0.384 ng/kg b.w. for adults and 0.559 ng/kg b.w. for the elderly. Values of EDI of AFM1 found in Londrina pose a toxicological risk to the population investigated. To the best of our knowledge, this is the first report on estimat ed AFM1 dietary exposure from Parana, Brazil.

Aflatoxina M1 (AFM1) e encontrada no leite e em outros produtos de excrecao apos o consumo de aflatoxina B1. AFM1 e carcinogenica para humanos, e avaliar os niveis de contaminacao em produtos lacteos e importante para conhecer os riscos aos quais a populacao esta exposta. A ocorrencia de AFM1 foi avaliada em 42 amostras de leite comercializadas em Londrina, Estado do Parana, Brasil, e sua ocorrencia foi utilizada para estimar sua exposicao. A determinacao de AFM1 foi avaliada por ELISA, e foi detectada em 100% das amostras, em niveis variando de 0,01 a 0,81 µ g/kg (media 0,13 µ g kg). Nenhuma das amostras apresentou niveis de AFM1 acima do maximo permitido pela Legislacao brasileira (0,5 µ g/kg para leite fluido e 5 µ g/kg para leite em po). A ingestao diaria estimada (IDE) de AFM1 foi avaliada, e a ingestao media foi de 0,468 ng/kg de peso corporal (p.c.)/dia para adolescentes, 0,384 ng/kg p.c./dia para adultos e 0,559 ng/kg p.c./dia para idosos. Valores de IDE de AFM1 encontrados em Londrina supoem um risco toxicologico para a populacao investigada. Do melhor do nosso conhecimento, este e o primeiro trabalho sobre a exposicao estimada de AFM1 do Parana, Brasil.

Toxigenic mycobiota and mycotoxins in shrimp feed / Micobiota toxígena e micotoxinas em ração de camarão

The objective of this study was to identify the toxigenic mycobiota and the occurrence of aflatoxins in shrimp feed products intended for shrimp cultivated in the coastal area of the state of Piauí, Brazil, in three farms ("A", "B" and "C"). The toxigenic capacity of the fungal species isolated was tested for aflatoxins (AF) and ochratoxin A production. The fungal counts of shrimp feed were similar for the "A" and "B" farms at all cultivation phases, collection sites, in closed and opened packages (1.33 to 2.66CFU g-1 log10 -1). The lowest fungal counts were found in feed from "C" farm (0.65CFU g-1 log10 -1) from closed packages. Thirty-four strains of Aspergillus were detected with a greater prevalence of A. flavus. Two strains produced B1, B2, G1 and G2 aflatoxins at concentrations from 0.39 to 0.42ng g-1; 0.18 to 0.27ng g-1; 1.78ng g-1 and 0.09ng g-1 respectively and were classified as atypical A. flavus. Two strains of A. niger aggregate were OTA producers. Fifteen samples (13.88%) presented AFB1 contamination at levels ranging from 0.25ng to 360ng g-1. This study demonstrates the presence of toxigenic fungi in shrimp feed used at different phases of cultivation and farms. Atypical strains of A. flavus were isolated which produced AF B1, B2, G1 and G2 in shrimp feeds. Only AFB1 was detected in the analyzed feed.

O objetivo deste estudo foi identificar a micobiota toxigênica e a incidência de aflatoxinas em rações comerciais para camarão cultivado no litoral do Estado do Piauí, Brasil, em três fazendas ("A", "B" e "C"). Foi realizada a capacidade toxigênica das espécies de fungos isolados e a produção de aflatoxinas (AF) e ocratoxina A (OTA). As contagens fúngicas da ração foram semelhantes nas fazendas "A" e "B" e em todas as fases de cultivo, locais de coleta e de embalagens fechadas e abertas (1,33-2,66UFC g-1 log10 -1). As mais baixas contagens de fungos foram encontradas nas rações de embalagens fechadas da fazenda "C" (0,65UFC g-1 log10 -1). Foram isoladas trinta e quatro cepas de Aspergillus com maior prevalência de A. flavus e duas linhagens eram produtoras de aflatoxinas B1, B2, G1 e G2 em concentrações 0,39-0,42ng g-1; 0,18-0,27ng g-1; 1,78ng g-1, e 0,09ng g-1, respectivamente, e foi classificado como A. flavus atípico, sendo necessária posteriormente a classificação filogenética desta cepa. Duas cepas de A. niger agregados eram produtoras de OTA. Quinze amostras de ração (13,88%) apresentaram contaminação AFB1 em níveis que variam de 0,25ng a 360ng g-1. Este estudo demonstra a presença de fungos toxigênicos em rações de camarão nas fazendas analisadas e nas diferentes fases de cultivo. Foram isoladas, em rações de camarões, cepas atípicas de A. flavus, produzindo AF B1, B2, G1 e G2. Apenas AFB1 foi detectada na ração analisada.

Reduction of aflatoxin B1 during tortilla production and identification of degradation by-products by direct-injection electrospray mass spectrometry (DIESI-MS) / Reducción de la aflatoxina B1 durante la producción de tortilla y la identificación de los productos de degradación por espectrometría de masas con ionización por electrospray de inyección-directa (DIESI-MS)

Objective. To determine the effect of pH, and exposure time over the inactivation of aflatoxin B1 (AFB1) during the tortilla making process as well as the degradative molecules generated. Materials and methods. Inactivation of AFB1 in maize-dough with alkaline pH and in alkaline methanolic solutions was determined by HPLC. Kinetics of time exposure of AFB1 in methanolic solution and the degradative products were analyzed by direct injection electrospray mass spectometry (DIESI-MS). Results. The alkaline pH of the maize-dough after nixtamalización between 10.2, and 30-40 minutes of resting at room temperature allows the 100% reduction of AFB1. DIESI-MS analysis of the extracts indicated the presence of two degradation molecules from AFB1. Conclusion. The alkaline pH of maize-dough and resting time are the principal factors involved in diminishing AFB1 levels in tortillas. A procedure to the tortilla making process is proposed, which allows the reduction of remnant AFB1, avoiding the accumulative effect over consumers.

Objetivo. Determinar el efecto del pH alcalino de la masa de maíz y el tiempo de exposición sobre la aflatoxina B1 (AFB1) durante la producción de tortillas e identificar los posibles productos de degradación mediante DIESI-MS. Material y métodos. La inactivación de la AFB1 a pH alcalino y diferentes tiempos de exposición en masa nixtamalizada y en soluciones metanólicas fueron determinadas por HPLC. La cinética de degradación de AFB1, y los productos de degradación en soluciones metanólicas se determinaron por DIESI-MS. Resultados. El pH alcalino de la masa y 30 a 40 minutos de reposo redujeron en 100% la AFB1 adicionada. Se identificaron dos moléculas de degradación. Conclusión. Los principales factores involucrados en la disminución de la AFB1 durante la producción de tortillas son la hidrólisis alcalina y el tiempo de reposo. Se propone un procedimiento para la producción de tortilla que reducirá la AFB1 residual evitando el efecto acumulativo en los consumidores.

Estudo sobre a ocorrência de fungos e aflatoxina B1 na dieta de bovinos leiteiros em São Paulo / Study on the occurrence of fungi and aflatoxina B1 in the diet of dairy cattle in São Paulo, Brazil

A qualidade da dieta ofertada às vacas em lactação é uma preocupação dos agentes de saúde devido à possibilidade da detecção de micotoxinas prejudiciais a saúde humana e animal. Os objetivos do trabalho foram avaliar o perfil da micobiota, determinar a atividade de água (Aa) e a ocorrência natural de aflatoxina B1 (AFB1) em dietas ofertadas a vacas em lactação de fazendas leiteiras no estado de São Paulo, Brasil. As amostragens das dietas foram realizadas diretamente dos cochos de lote de 15 vacas, em dois dias consecutivos com intervalos de 24h e a cada 15 dias, perfazendo um período de 45 dias de amostragens por fazenda. A purificação e determinação de AFB1 foram realizadas em colunas de imunoafinidade e Cromatografia Líquida de Alta Eficiência (CLAE). O estudo da micobiota presente nas amostras das dietas (288) revelou que as leveduras foram predominantes em todas as dietas (83,97 a 99,98%). Foram isolados 15 gêneros de fungos filamentosos, com os gêneros Aspergillus spp (20,09%), Fusarium spp (14,16%) e Penicillium spp (11,48%) os mais prevalentes. As contagens de Unidades Formadoras de Colônias por grama de alimento (UFC. g-1) variaram de 102 a 1011. A atividade de água das amostras variou entre 0,91 a 0,98. Foi detectada a presença de AFB1 em 31,44% das amostras com teores entre 1,68 a 194,51μg.kg-1. Medidas de boas práticas de produção, estocagem e utilização devem ser tomadas para diminuir a ocorrência de AFB1 nas dietas ofertadas às vacas em lactação...

The quality of the diet offered to lactating cows is a concern to health officials the possibility of detecting mycotoxins harmful to human and animal health. The objectives were to evaluate the profile of mycoflora, determine the water activity (Aw) and the natural occurrence of aflatoxin B1 (AFB1) in diets offered to lactating cows from dairy farms in the state of São Paulo, Brazil. Samples of the diets were taken directly from the troughs batch of 15 cows, on two consecutive days at intervals of 24 hours and every 15 days with a period of 45 sampling days per farm. Purification and determination of AFB1 were performed on immunoaffinity columns and High Performance Liquid Chromatography (HPLC). The study of mycobiota present in samples of diets (288) revealed that yeast cells were predominant in all diets (83.97 to 99.98%). 15 genera were isolated from filamentous fungi, with Aspergillus spp (20.09%), Fusarium spp. (14.16%) and Penicillium spp. (11.48%) the most prevalent. The counts of colony forming units per gram of food (UFC.g-1) ranged from 102 a1011. The water activity of the samples ranged from 0.91 to 0.98. We have detected the presence of AFB1 in 31.44% of samples with levels between 1.68 a 194.51μg.kg-1. Measures of good production, storage and use should be taken to reduce the occurrence of aflatoxin B1 in the diet offered to lactating cow...

Niveles de Ocratoxina A y Aflatoxinas totales en cafés de exportación de Panamá por un método de ELISA

Se realizó un estudio de las condiciones del procesamiento del café de exportación en 15 beneficios, ubicados en Chiriquí, región occidental de Panamá. Además se analizaron 21 muestras de café procesado (grano verde), provenientes de los beneficios. Las muestras fueron analizadas microbiológicamente y se cuantificaron las Aflatoxinas totales (B1, B2, G1 y G2) y Ocratoxina A (OTA), mediante el método de inmunoafinidad ELISA. Se determinó un límite de detección de 0,017 ng/mL, para la Ocratoxina A, lo que equivale a una concentración de 0,829 μg/kg en la muestra, y un límite de detección de 0,027 ng/mL, para las Aflatoxinas totales, lo que equivale a una concentración de 1,350 μg/kg de Aflatoxinas totales. En la muestra, se encontró que cuatro de las 21 (19%) resultaron positivas a la presencia de Ocratoxina A y tres, a la presencia de Aflatoxinas totales (14%). Las muestras presentaron niveles de Ocratoxina A en el rango de 4,90-37,73 μg/kg; sólo tres de ellas superaron el límite máximo permitido por la Unión Europea, para la concentración de Ocratoxina, que es de 5,0 μg/kg. Las Aflatoxinas totales se encontraron en el rango de 1,51- 1,93 μg/kg, por debajo de los 10 μg/kg, que es el límite máximo permitido en el café por la Unión Europea. Los resultados nos indican que el procesamiento de café producido en Panamá cumple satisfactoriamente con los estándares internacionales de manejo poscosecha, lo que conduce a una baja incidencia de hongos productores de micotoxinas y niveles muy bajos de micotoxinas.

Levels of Ochratoxin A and total Aflatoxins in Panamanian exportation coffee by an ELISA Method. A study about processing conditions of exportation coffee in 15 benefits located in Chiriquí, western region of Panama, was conducted. In addition, 21 samples of processed coffee (green beans), from the benefits, were analyzed. The samples were microbiologically tested in order to quantify total aflatoxins (B1, B2, G1 and G2) and Ochratoxin A (OTA), using the immunoaffinity ELISA method. A detection limit of 0.017 ng/mL, was determined for Ochratoxin A, which is equivalent to a concentration of 0.829 μg/kg, and a detection limit of 0.027 ng/mL, for total aflatoxins, which is equivalent to a concentration of 1.350 μg/kg. It was found that four (19%) out of the 21 samples were positive to the presence of Ochratoxin A and three (14%) to the presence of total aflatoxins. Samples showed levels of Ochratoxin A in the range 4.90 - 37.73 μg/kg; only three of them exceeded the maximum limit allowed by the European Union, for the concentration of Ochratoxin, which is of 5.0 μg/kg. Total aflatoxins were found in the range 1.51 - 1.93 μg/kg, below 10μg/kg which is the maximum limit allowed for coffee by the European Union. The results indicate that the processing of coffee produced in Panama successfully meets international standards for postharvest handling, which leads to a low incidence of mycotoxins and very low levels of mycotoxin- producing fungi.

Exposición a aflatoxina: un problema de salud pública / Exposition to aflatoxin: A public health problem

La aflatoxina, una micotoxina producida por hongos contaminantes, es un potente tóxico hepático y un agente carcinógeno. La exposición a ella en la dieta es de particular importancia en ciertas regiones del Sureste de Asia y de África subsahariana, cuyas poblaciones presentan alta frecuencia de carcinoma hepatocelular y de la mutación en el codón 249 del gen p53; además, tienen alta prevalencia de la infección por el virus de la hepatitis B. Este factor de riesgo es muy importante si se tiene en cuenta que se ha demostrado sinergia entre la infección por dicho virus y la exposición a aflatoxina en la patogénesis del carcinoma hepatocelular. Pocos estudios han explorado la exposición a aflatoxinas en la dieta de la población latinoamericana y se desconoce el papel en ella de esta micotoxina como factor de riesgo para dicho carcinoma. En este artículo se presenta una revisión sobre diversos aspectos de las aflatoxinas, con énfasis en su relación con la infección por el virus de la hepatitis B y con el carcinoma hepatocelular.

Aflatoxin, a mycotoxin produced by pollutant molds, is a potent hepatotoxic and carcinogenic agent. Dietary exposition to it is of particular importance in certain regions of Southeast Asia and sub-Saharan Africa. Populations in these regions suffer from high incidence of hepatocellular carcinoma, and have high frequency of the mutation in the codon 249 of p53 gene; besides, prevalence of Hepatitis B virus (HBV) infection is high in those populations. Synergism between infection with HBV and the exposition to this mycotoxin in the pathogenesis of hepatocellular carcinoma has been demonstrated. Few studies have explored the exposition to aflatoxin in the diet of populations in Latin America, and the role in them of this mycotoxin as a risk factor for hepatocellular carcinoma is unknown. In this article different aspects of aflatoxin are reviewed with emphasis on its relationship with HBV infection and with such neoplasia.

Análise micológica e toxicológica em amendoim e derivados / Mycological and toxicological analysis of peanuts and derivatives

The filamentous fungi Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius produce toxins, which are secondary metabolites called aflatoxins. These toxin-producing species grow rapidly on peanuts and cereals in favorable conditions of temperature and humidity. Their toxins can either cause acute effects, and even be lethal or accumulate in the organism, resulting in liver cancer in the long term. Based on the health risks of aflatoxins in food, a research was conducted on peanuts and derivatives sold in Alfenas, MG, Brazil, to evaluate the presence of Aspergillus sp. and aflatoxins. The samples were randomly collected at ?popular stores?, from November 2008 to May 2009, to assess the occurrence of aflatoxins B1, B2, G1 and G2 by thin layer chromatography (TLC). The mycological analysis revealed the presence of fungi in 50% of the samples: Penicillium sp. (53.85%), A. flavus (19.23%), A. niger (15.38%) and A. fumigatus (11.54%), and 63.64% of these showed the presence of aflatoxins : B1 (43.14%), B2 (25.49%), G1 (23.53%) and G2 (7.84%). It is concluded that the results reported here are a cause for concern, given the harmfulness of these cumulative and carcinogenic toxins.

As espécies Aspergillus flavus, Aspergillus parasiticus e Aspergillus nomius produzem micotoxinas, que são metabólitos secundários denominados de aflatoxinas. Desenvolvem-se rapidamente em amendoins e cereais produzindo esta toxina, quando em condições favoráveis de temperatura e umidade. Essas toxinas podem produzir efeitos agudos, letais ou não, ou efeito cumulativo no organismo, que a longo prazo, podem causar, principalmente, lesões carcinogênicas hepáticas. Sabendo-se dos riscos à saúde, devido à presença de aflatoxinas nos alimentos, foi realizada uma pesquisa em amendoins e derivados comercializados na cidade de Alfenas ? MG, Brasil, para avaliar a presença de aflatoxinas e de fungos do gênero Aspergillus sp. Amostras foram coletadas aleatoriamente em lojas populares no período de novembro de 2008 a maio de 2009 e avaliada a ocorrência de aflatoxinas B1, B2, G1 e G2, utilizando-se a técnica de Cromatografia de Camada Delgada (CCD). Na análise micológica foi constatada a presença de fungos em 50% das amostras, das espécies: Penicillium sp. (53,85%), A. flavus (19,23%), A. niger (15,38%), A. fumigatus (11,54%). Obteve-se como resultado para a presença de aflatoxinas 63,64 %, sendo: B1 (43,14%), B2 (25,49%), G1 (23,53%), G2 (7,84%). Concluiu-se que os números encontrados são preocupantes, tendo em vista a periculosidade desta substância

Mecanismos moleculares involucrados en la mutagenicidad inducida por aflatoxina B1 / Molecular mechanism involved in the mutagenicity induced by Aflatoxin B1 / Mecanismos moleculares envolvidos na mutagenicidade induzida pela aflatoxina B1

La aflatoxina B1 (AFB1) es una micotoxina identificada como el más potente hepatocarcinógeno. El metabolito que resulta del proceso de detoxificación de la AFB1 en el hígado tiene la capacidad de reaccionar con el ADN genómico, generando el aducto AFB1-ADN; durante la replicación del ADN este aducto induce la transversión G:C—>T:A. Polimorfismos en los genes que codifican las enzimas encargadas de la activación y detoxificación de la AFB1 y enzimas de reparación del ADN han sido asociados con el riesgo de desarrollar carcinoma hepatocelular (CHC). Adicionalmente, en poblaciones con alta exposición a aflatoxina y alta prevalencia de infección por el virus de la hepatitis B (VHB) se ha demostrado un sinergismo entre estos dos factores de riesgo para el desarrollo de CHC.

The aflatoxin B1 (AFB1) is a mycotoxin that has been identified as the most potent hepatocarcinogen. The metabolite resulting from detoxification process of AFB1 in liver, has the ability to react with the genomic DNA, generating AFB1-DNA adducts; during DNA replication process, this adduct induced the G:C—>T:A transversion. Polymorphism in genes encoding for enzymes involved in the activation and detoxification of AFB1 and DNA repair enzymes has been associated with the risk of hepatocellular carcinoma (HCC) development. Additionally, in populations of high exposure to aflatoxin and high prevalence of hepatitis B virus (HBV) infection, has been demonstrated a synergism between these two risk factors for the development of HCC.

Aflatoxina B1 (AFB1) é uma micotoxina identificado como o hepatocarcinogen mais potente. O metabolito resultante do processo de desintoxicação de AFB1 no fígado, tem a capacidade de reagir com o ADN genómico, gerando AFB1 DNA-aducto; transversão durante a replicação do ADN deste aducto induz G:C—>T:A. Polimorfismos em genes que codificam as enzimas envolvidas na activação e na desintoxicação de AFB1 e enzimas de reparação do ADN têm sido associados com o risco de desenvolvimento de carcinoma hepatocelular (HCC). Além disso, em populações com elevada exposição a aflatoxina e elevada prevalência da infecção com vírus da hepatite B (VHB) tem sido mostrado um sinergismo entre estes dois factores de risco para o desenvolvimento de carcinoma hepatocelular.

Intoxicação experimental por aflatoxina em bezerros / Experimental aflatoxin poisoning in calves

Foram realizados dois experimentos para determinar os efeitos tóxicos de diferentes doses de aflatoxinas em bezerros, considerando-se aspectos clínicos, produtivos e patológicos. ... No primeiro experimento, o ganho de peso dos bezerros recebendo AFB1 foi equivalente ao do grupo controle durante todo período experimental. ...Durante o período experimental, e três semanas após o término desse período, não foram observados sinais clínicos e alterações histopatológicas associadas ao consumo de aflatoxinas, em qualquer dos bezerros do grupo tratamento do primeiro experimento.... Níveis alterados da atividade sérica de FA e GGT foram observados em todos os bezerros do grupo tratamento durante grande parte do período experimental. Queda acentuada do nível da AS sérica foi observada na coleta do 49º dia do experimento no bezerro que recebia a maior dose de aflatoxina. Não foram observadas variações no hematócrito e na atividade sérica da AST, nem nos níveis séricos de proteína total, bilirrubina total e bilirrubina direta em qualquer dos bezerros desse experimento. Alterações histopatológicas nos bezerros intoxicados incluíram proliferação de ductos biliares, degeneração citoplasmática vacuolar consistente com acumulação hepatocelular de lipídios, fibrose periportal, ou em ponte, megalocitose, fibrose subendotelial das veias hepáticas terminais e edema. Achados de necropsia do bezerro recebendo a maior dose de AFB1 incluíram fígado levemente aumentado de tamanho, difusamente amarelo-claro e firme, discreta ascite, edema de mesentério e submucosa do abomaso. Os dados obtidos nesses experimentos permitem afirmar que doses de 500±100 ppb de AFB1 não causam alterações patológicas e produtivas em bezerros em condições experimentais, mas podem estar associadas à mínimas alterações bioquímicas, enquanto doses de 1.250, 2.500 e 5.000 ppb de aflatoxina B1 causam doença hepática crônica em bezerros em condições experimentais.

Two experiments were performed in order to determine the toxic effects of varying doses of aflatoxins in calves. Clinical, productive and pathologic aspects of affected calves were considered….During all the experimental period of the first experiment, the weight gain of the calves receiving AFB1 was equivalent to that of the control group.…During the whole experimental period and up to three weeks after the final of the experiment, no clinical signs or histopathological changes associated with the consumption of aflatoxins were observed in any of the calves of the first experiment….Increased activity of AF and GGT were observed in all the calves of the treated group during most part of the experimental period. A marked drop in the serum levels of SA was observed in the serum sampled on the 49º day of the experiment in the calf receiving. the largest dose of aflatoxin. No changes were observed regarding PCV, TP, total bilirubin, direct bilirubin and in the serum activity of AST in any of the calves of the second experiment. Histopathological changes in intoxicated calves included bile duct proliferation, cytoplasmic vacuolar hepatocelular degeneration consistent with hepatocelular deposit of lipids, periportal to bridging fibrosis, megalocytosis, subendothelial edema and fibrosis in terminal hepatic veins. Necropsy findings in the euthanatized calf which receive de largest doses of AFB1 included slight enlargement of the liver which was firm and diffusely light-yellow, mild ascites, and edema of the mesentery and of abomasal folds. Data stemmed from these two experiments allow to conclude that AFB1 doses of 500±100 in the ration do not cause pathologic changes or decrease in productivity in calves kept in experimental conditions, but can be associated to minimal serum biochemistry; while AFB1 doses of 1.250, 2.500 e 5.000 ppb in the ration cause chronic hepatic disease in calves in kept in experimental conditions.

Qualidade da castanha-do-brasil do comércio de Rio Branco, Acre / Quality of Brazil nuts marketed in Rio Branco, Acre

Este trabalho teve como objetivo avaliar a qualidade de castanhas-do-brasil beneficiadas e comercializadas em Rio Branco, Acre. Foram analisadas amostras das três marcas de castanha encontradas no mercado local quanto às variáveis: atividade de água, teor de umidade, contagem total de fungos filamentosos, quantificação de Aspergillus flavus e de A. parasiticus, bem como quantificação de aflatoxinas B1, B2, G1 e G2. As castanhas do comércio se encontravam com um teor de umidade e atividade de água adequados, o que pode ter sido responsável pela baixa contaminação por fungos e por aflatoxinas. Quanto a estas micotoxinas, as amostras estão de acordo com o recomendado pela Anvisa, podendo ser esta uma consequência da grande divulgação no Estado do uso de Boas Práticas no manejo da castanha.

The goal of this paper was to evaluate the quality of Brazil nuts processed and marketed in the city of Rio Branco, in the state of Acre (Brazil). We analysed three samples for water activity, moisture content, total fungus quantification of Aspergillus flavus and A. parasiticus, as well as quantification of total aflatoxin, Afla B1, Afla B2, Afla G1 and Afla G2. The nut samples from the market showed an appropriate moisture content and water activity, which may have been responsible for the low fungus contamination and aflatoxin production. As to these mycotoxins, the samples were consistent with Anvisa's recommendations, which may be a consequence of good management of the nuts in Acre.