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.

Influence of a glyphosate-based herbicide on growth parameters and aflatoxin B1 production by Aspergillus section Flavi on maize grains.

Glyphosate-based herbicides (GBH) are the main pesticides applied worldwide on maize production. Glyphosate-resistant weeds led to the repeated application of high doses of the pesticide. In addition to environmental conditions, the presence of GBH affects the development of Aspergillus species and aflatoxin B1 (AFB1) production under in vitro conditions. The aim of this work was to evaluate the influence of a commercial GBH on growth and AFB1 production by Aspergillus flavus and Aspergillus parasiticus strains under different water activity (aW) conditions. The following concentrations of active ingredient glyphosate were evaluated: 20, 50, 200 and 500mM. The lag phase prior to growth and growth rate did not change at 20 and 50mM (that is, at field recommended doses) at 0.98 and 0.95 aW; however, at increasing GBH concentrations, between 200 and 500mM, the growth rate decreased at all aW conditions. In general, as the GBH concentration increased, AFB1 production decreased. However, a significant increase in toxin accumulation was found only at one of the aW conditions (0.95) at 21 days with 50mM of GBH in A. flavus and 20 and 50mM of GBH in A. parasiticus. These results show that, even though Aspergillus section Flavi growth did not increase, AFB1 production increased on maize grains at GBH concentrations similar to those of field recommended doses under favorable water availability and temperature conditions.

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)

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.

Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver.

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=0.050ng/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.

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.

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 (aw, 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 aw 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.

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...

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.

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.

Evaluation of antifungal activity of Pittosporum undulatum L. essential oil against Aspergillus flavus and aflatoxin production / Avaliação da atividade antifúngica do óleo essencial de Pittosporum undulatum L. em Aspergillus flavus e produção de aflatoxina

The presence of mycotoxins as a result of fungal attack can occur before, after and during the harvest and storage operations on agricultural crops and food commodities. Considering the inhibitory property of essential plant oils on the mycelial development of fungi and the importance of Aspergillus flavus, the main producer of aflatoxins, this research was designed to evaluate the toxicity of essential oil from Pittosporum undulatum against A. flavus. The essential oils were obtained from P. undulatum leaves, collected in different months and analyzed by GC/MS. The oils were rich in hydrocarbon, monoterpenes and sesquiterpenes and it was observed a significant variation on the chemical composition of the essential oil of leaves at different months. Besides, the essential oils were tested against fungal growth and the results showed different spectrum of inhibition on A. flavus. However, the essential oils inhibited the aflatoxin B1 production.

A presença de micotoxinas como resultado do ataque fúngico pode ocorrer antes, após e durante a colheita e também no armazenamento de grãos e alimentos. Considerando as propriedades inibitórias dos óleos essenciais de plantas no desenvolvimento do micélio dos fungos e a importâncias do Aspergillus flavus, principal produtor de aflatoxinas, relatou-se neste trabalho, a atividade tóxica do óleo essencial do Pittosporum undulatum em cultura de A. flavus. Os óleos essenciais de P. undulatum foram obtidos a partir de folhas coletadas em diferentes meses e analisado por CG/EM. Os óleos se mostraram ricos em hidrocarbonetos, monoterpenos e sesquiterpenos e foi observada uma significante variação na composição química destes óleos nos diferentes meses de coleta. Os óleos essenciais mostraram diferentes espectros de inibição do crescimento de A. flavus, porém todos foram capazes de inibir a produção de aflatoxina B1.

Effect of gamma radiation on the inactivation of aflatoxin B1 in food and feed crops / Efeito da radiação gama na inativação de aflatoxina B1 em alimentos e ração

Samples of food crops (peanut, peeled pistachio, unpeeled pistachio, rice, and corn) and feed (barley, bran, corn) were autoclave-sterilized, and inoculated with 10(6) of spore suspension of an isolate of Aspergillus flavus fungus known to produce aflatoxin B1 (AFB1) . Following a 10-day period of incubation at 27 C to allow for fungal growth, food and feed samples were irradiated with gamma radiation at the doses 4, 6, and 10 kGy. Results indicated that degradation of AFB1 was positively correlated with the increase in the applied dose of gamma ray for each tested sample. At a dose of 10 kGy percentages of AFB1 degradation reached highest values at 58.6, 68.8, 84.6, 81.1 and 87.8 percent for peanuts, peeled pistachios, unpeeled pistachios, corn and rice samples, respectively. In feed samples percentages of AFB1 degradation were 45, 66, and 90 percent in barley, 47, 75, and 86 percent in bran, and 31, 72, and 84 percent in corn for the doses of 4, 6, and 10 kGy, respectively. AFB1 degradation in food samples correlated negatively with oil content in irradiated samples. Thus, in peanuts, which contained the highest oil content, percentage of AFB1 degradation at 10 kGy was not more than 56.6 percent, whereas, the corresponding value in corn, which contained the lowest oil content, reached as high as 80 percent. The above results indicate the possibility of using gamma radiation as a means of degradation of AFB1 in food and feed crops to levels lower than the maximum allowed levels.

Amostras de alimentos (amendoim, pistache descascada, pistache com casca, arroz e milho) e de ração (cevada, farelo de trigo e milho) foram esterilizadas por autoclavação e inoculadas com uma suspensão de esporos (10(6)) de um isolado de Aspergillus flavus produtor de aflatoxina B1 (AFB1). Após incubação por 10 dias a 27ºC para multiplicação do fungo, as amostras foram irradiadas com radiação gama nas doses de 4, 6 e 10 kGy. Os resultados indicaram que a degradação da AFB1 correlacionou-se positivamente com o aumento da dose de radiação gama. As porcentagens de degradação da AFB1 foram mais altas na dose de 10kGy, obtendo-se valores de 58,6, 68,8, 84,6, 81,1 e 87,8 por cento para amendoim, pistache descascada, pistache com casca, milho e arroz, respectivamente. Nas rações, as porcentagens de degradação de AFB1 foram 45, 66 e 90 por cento para cevada, 47, 75 e 86 por cento para farelo de trigo e 31, 72 e 84 por cento para milho, nas doses de 4, 6 e 10 kGy, respectivamente. A degradação de AFB1 correlacionou-se negativamente com o teor de gordura nas amostras irradiadas. Assim, em amendoim, que apresentou o teor de gordura mais alto, a porcentagem de degradação com 10 kGy foi inferior a 56,6 por cento, enquanto o valor correspondente em milho, que apresentou o teor de gordura mais baixo, foi de 80 por cento. Os resultados indicam a possibilidade de uso da radiação gama como meio de degradação de AFB1 em alimentos e ração a níveis inferiores ao máximo permitido.

Umbrales para las clases específicas de carcinógenos genotóxicos. nueva estrategia para la categorización de la carcinogenicidad de las sustancias químicas / Thresholds for specific classes of genotoxic carcinogens: a new strategy for carcinogenicity categorisation of chemicals

Cada vez está más claro el principio general de la secuencia de acontecimientos que finalmente conducen al cáncer después de la exposición a carcinógenos genotóxicos. Esto ayuda a conocer los parámetros que influyen en la forma de la curva dosis-efecto para la carcinogénesis, que incluyen activación e inactivación metabólica de carcinógenos, reparación del ADN, control del ciclo celular, proliferación regenerativa, apoptosis, senescencia inducida por oncogenes y control por el sistema inmunitario. Una relación lineal dosis-respuesta sin umbral observable parece ser una descripción conservadora pero adecuada para la actividad carcinógena de muchos carcinógenos genotóxicos, por ejemplo, la aflatoxina B1. Sin embargo, algunos modelos de extrapolación lineal que conectan el riesgo de alto nivel a la intersección en el cero han conducido a predicciones erróneas. En esta revisión se demuestra que el acetato de vinilo es un ejemplo de carcinógeno que actúa a través de un mecanismo de umbral. En los tejidos de contacto, el acetato de vinilo es convertido en ácido acético y acetaldehído. Sólo cuando se alcanzan las concentraciones umbral se activa el mecanismo que finalmente conduce al cáncer, es decir una reducción del pH mayor de 0.15 unidad que conduce a citotoxicidad, daño del ADN y proliferación regenerativa. En esta revisión se presenta un nuevo sistema de categorización de los carcinógenos que tiene en cuenta que pueden actuar por mecanismos de umbral.

The general principle of the sequence of events that finallylead to cancer after exposure to genotoxic carcinogenshas become increasingly clear. This helps to understandthe parameters that influence the shape of the dose effectcurve for carcinogenesis, including metabolic activationand inactivation of carcinogens, DNA repair, cell cyclecontrol, regenerative proliferation, apoptosis, oncogeneinducedsenescence and control by the immune system.A linear dose response relationship with no observablethreshold seems to be a conservative but adequatedescription for the carcinogenic activity of many genotoxiccarcinogens, such as for instance aflatoxin B1. However,some linear extrapolation models connecting the highlevelrisk to the zero intercept have resulted in wrongpredictions. In this review we demonstrate that vinylacetate is an example of a carcinogen acting by athreshold mechanism. In tissues of contact vinyl acetateis converted to acetic acid and acetaldehyde. Only whenthreshold levels are achieved critical steps in themechanism that ultimately leads to cancer become active,namely pH reduction of more than 0.15 units leadingto cytotoxicity, damage to DNA and regenerativeproliferation. In this review we present a new system ofcarcinogen categorisation taking into account thatcarcinogens may act by threshold mechanisms.

Effect of gamma irradiation on the inactivation of aflatoxin B1 and fungal flora in peanut

The effect of gamma irradiation on aflatoxin B1 levels and fungal infection were investigated in peanut samples, Tatu Vermelho cultivar. At a radiation dose of 10 KGy, growth of molds was completely inhibited. Doses of 15, 20, 25 and 30 KGy were sufficient for destruction of aflatoxin B1 by 55-74%. The results suggested that the decontamination of molds by irradiation, before production of aflatoxin B1, is the most acceptable method in the preservation of peanut.

O efeito da irradiação gama nos níveis de aflatoxina B1 e na infecção fúngica foram investigadas em amostras de amendoim, cultivar Tatu Vermelho. Dose de irradiação gama (60Co) de 10 KGy inibiu completamente o crescimento de fungos. Doses de 15, 20, 25 e 30 KGy foram suficientes para destruição de aflatoxina B1 de 55 a 74%. Pode-se concluir do presente trabalho, que a descontaminação de fungos por irradiação gama antes da produção de aflatoxina B1 é o método apropriado na preservação de amendoim.

Effect of gamma irradiation on the inactivation of aflatoxin B1 and fungal flora in peanut

The effect of gamma irradiation on aflatoxin B1 levels and fungal infection were investigated in peanut samples, Tatu Vermelho cultivar. At a radiation dose of 10 KGy, growth of molds was completely inhibited. Doses of 15, 20, 25 and 30 KGy were sufficient for destruction of aflatoxin B1 by 55-74%. The results suggested that the decontamination of molds by irradiation, before production of aflatoxin B1, is the most acceptable method in the preservation of peanut.

O efeito da irradiação gama nos níveis de aflatoxina B1 e na infecção fúngica foram investigadas em amostras de amendoim, cultivar Tatu Vermelho. Dose de irradiação gama (60Co) de 10 KGy inibiu completamente o crescimento de fungos. Doses de 15, 20, 25 e 30 KGy foram suficientes para destruição de aflatoxina B1 de 55 a 74%. Pode-se concluir do presente trabalho, que a descontaminação de fungos por irradiação gama antes da produção de aflatoxina B1 é o método apropriado na preservação de amendoim.

Ocorrência de aflatoxina B1 em produtos alimentícios e rações animais, consumidos no Estado de Säo Paulo e em várias outras regiões do Brasil no período de 1980 a 1987 / Incidence of aflatoxin B1 in foodstuffs and animal feed, consumed in Säo Paulo and in various areas of Brazil

Aflatoxina B1 foi identificada e quantificada por cromatografia em camada delgada em 666 amostras de produtos alimentícios e 308 amostras de rações animais expostas ao consumo no Estado de São Paulo e em várias outras regiões do Brasil. Foi detectada aflatoxina B1 em 3,39% do total das amostras analisadas, com concentração superior a 30 mg/kg(ppb), que é o limite tolerado pela legislação brasileira. Os resultados foram expressos em tabelas (AU).