Detección de siete micotoxinas tricotecenos en cereales infantiles mediante extracción QuEChERS y cromatografía líquida espectrometría de masas en tándem / Detection of seven trichothecene mycotoxins in infant cereal foods by QuEChERS extraction and liquid chromatography coupled to tandem mass spectrometry

Las micotoxinas tricotecenos se encuentran comúnmente en los cereales, como el trigo, la cebada, el maíz, la avena, el centeno y los productos derivados. Los bebés y los niños pequeños se consideran un grupode alto riesgo debido al gran consumo de alimentos a base de cereales en relación con su peso corporal. En este estudio se ha desarrollado y validado un método rápido, selectivo y sensible para la cuantificación simultánea de 7 micotoxinas tricotecenos (HT-2 toxina, T-2, diacetoxiscirpenol (DAS), deoxinivalenol (DON), 3-acetil-DON, 15-acetil DON, y fusarenon-X (FUS-X) en cereales infantiles. Las micotoxinas se han extraído de las muestras mediante QuEChERS modificado (acrónimo de rápido, fácil, barato, eficaz, robusto y seguro). El método se basa en una única extracción con una mezcla de metanol y acetonitrilo, seguido de una etapa de extracción/partición después de la adición de la sal, y un paso de limpieza utilizando extracción en fase sólida de dispersión (EPS-D). El análisis se llevó a cabo usando cromatografía líquida combinada con un espectrómetro de masas en tándem de triple cuadrupolo (EM/EM) con ionización por electrospray en modo positivos (ESI+) con monitorización de reacción múltiple (MRM). El método validado demostró tener una buena exactitud (>72% de recuperación), reproducibilidad (<10% de desviación estándar relativa interdia) y sensibilidad para las micotoxinas seleccionadas (los límites de detección del método oscilaron entre 0,02 mg kg-1 a 0,15 mg kg-1). Se analizaron 57 muestras de cereales infantiles comercializadas en España revelando la presencia de FUS-X, 15-acetil DON, 3-acetil-DON y T-2 en 9 (15,5%) muestras (AU)

Trichothecene mycotoxins are commonly found in cereals, including wheat, barley, maize, oats, rye and derived products. Infants and young children are considered the highest risk group because their large dietary intake of cereal-based foods in relation to their body weight. In this study, a rapid, selective and sensitive method was developed and validated for the simultaneous quantification of 7 trichothecenes (HT-2 toxin, T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON, and fusarenon-X (FUS-X) in infant cereal food. Mycotoxins have been extracted from the samples using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure. The method was based on a single extraction with a mixture methanol and acetonitrile, followed by an extraction/partitioning step after the addition of salt, and a cleanup step utilizing dispersive solid-phase extraction (D-SPE). The analysis was carried out using a liquid chromatography combined with a triple–quadrupole tandem mass spectrometer (MS/MS) by electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). The validated method showed to be accurate (>72% recovery), reproducible (<10% relative standard deviation interday) and sensitive for the selected mycotoxins (method detection limits ranged from 0.02 mg kg-1 to 0.15 mg kg-1). The screening of 57 samples of infant cereal food commercialized in Spain revealed the presence of FUS-X, 15-Acetyl-DON, 3-Acetyl- DON and T-2 in 9 (15.5%) samples (AU)

Is Penicillium citrinum implicated in sago hemolytic disease?

Sago hemolytic disease (SHD) is an acute hemolytic syndrome affecting rural Papua New Guineans who depend on the starch of Metroxylon sagu as a staple carbohydrate. It is a suspected mycotoxicosis associated with fungal succession in stored and perhaps poorly fermented sago. Despite a mortality rate of approximately 25%, little is know about the disease. Recent studies have identified Penicillium citrinum as a possible candidate in the etiology of SHD. This is based on the frequency of isolation from sago starch and the hemolytic nature of the organism as demonstrated when cultured on sheep and human blood agar. A highly non-polar lipophilic P. citrinum fraction from C18 solid phase extraction demonstrated high hemolytic activity in a semi-quantitative assay using both mouse and human erythrocytes. When the red cell membrane proteins were subjected to sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, cleavage of protein band 3 and spectrin was demonstrated. This breach of major structural red cell proteins is consistent with the severe hemolysis found in vivo. Our findings warrant further investigation into the hemolytic activity of P. citrinum and its role as the etiological agent of SHD.

Molds, mycotoxins, and nutrition.

Many of the patients with mold and chemical exposure that come to Environmental Health Center - Dallas (EHC-D) show signs of impaired digestion. Improving their digestion improves their nutritional status and also improves their detoxification abilities. Mold and chemical exposure also affect the patient's short-term memory and executive function. This presents a challenge when it comes to teaching the patient how to improve their nutritional status and detoxification. At the EHC-D, we use several instructions methods that have resulted in the patient's grasping the concepts and then being able to implement the required changes.

Do terbutaline- and mold-associated impairments of the brain and lung relate to autism?

Increased prevalence of the autism spectrum disorders (ASD) and the failure to find genetic explanations has pushed the hunt for environmental causes. These disorders are defined clinically but lack objective characterization. To meet this need, we measured neurobehavioral and pulmonary functions in eight ASD boys aged 8 to 19 years diagnosed clinically and compared them to 145 unaffected children from a community with no known chemical exposures. As 6 of 35 consecutive mold/mycotoxin (mold)-exposed children aged 5 to 13 years had ASD, we compared them to the 29 non-ASD mold-exposed children, and to the eight ASD boys. Comparisons were adjusted for age, height, weight, and grade attained in school. The eight ASD boys averaged 6.8 abnormalities compared to 1.0 in community control boys. The six mold-exposed ASD children averaged 12.2 abnormalities. The most frequent abnormality in both groups was balance, followed by visual field quadrants, and then prolonged blink reflex latency. Neuropsychological abnormalities were more frequent in mold-exposed than in terbutaline-exposed children and included digit symbol substitution, peg placement, fingertip number writing errors, and picture completion. Profile of mood status scores averaged 26.8 in terbutaline-exposed, 52 in mold exposed, and 26 in unexposed. The mean frequencies of 35 symptoms were 4.7 in terbutaline, 5.4 in mold/mycotoxins exposed and 1.7 in community controls.

Presumptive tremorgenic mycotoxicosis in a dog in New Zealand, after eating mouldy walnuts.

CASE HISTORY: A 1-year-old, intact male Labrador-cross dog vomited after eating walnuts that had been on the ground for 5 months. The dog then developed tremors, ataxia, increased salivation, and hyperaesthesia. CLINICAL FINDINGS: The dog had marked generalised tremors, ataxia and a temperature of 39.9 degrees C. Both pupils were of normal size and normally responsive to light. Vomiting was induced, and walnut shell was visible in the vomitus. DIAGNOSIS: Due to the sudden onset of tremors, lack of exposure to other convulsive toxins, and the evidence of ingestion of walnuts, the provisional diagnosis was tremorgenic mycotoxicosis. The dog was treated symptomatically, and made a full recovery over 18 hours. Tremorgenic mycotoxins were detected within walnuts collected from the dog's environment. CLINICAL RELEVANCE: Fungi that produce tremorgenic mycotoxins are present in New Zealand. Intoxication should be suspected in dogs that suddenly develop muscle tremors, especially if there is a history of ingestion of mouldy food 2-3 hours prior to the development of tremors.

Expression profiling of heat stress effects on mice fed ergot alkaloids.

Fescue toxicosis affects wild and domestic animals consuming ergot alkaloids contained in tall fescue forage infected with the endophytic fungus, Neotyphodium coenophialum. When animals are consuming infected fescue (E+) forage during periods of elevated ambient temperatures (summer), a range of phenotypic disorders collectively called summer slump is observed. It is characterized by hyperthermia, with an accompanying decrease in feed intake, growth, milk yield, and reproductive fitness. Laboratory mice also exhibit symptoms of fescue toxicosis at thermoneutral (TN) temperature, as indicated by reduced growth rate and reproductive fitness. Our goal was to characterize the differences in gene expression in liver of mice exposed to summer-type heat stress (HS) and E+ when compared to mice fed E+ at TN temperature. Mice were fed E+ diet under HS (34 +/- 1 degrees C; n = 13; E+HS) or TN conditions (24 +/- 1 degrees C; n = 14; E+TN) for a period of 2 weeks between 47 and 60 days of age. Genes differentially expressed between E+HS versus E+TN were identified using DNA microarrays. Forty-one genes were differentially expressed between treatment groups. Expressions of eight genes were measured using quantitative real-time PCR. Genes coding for phase I detoxification enzymes were upregulated in E+HS mouse liver. This detoxification pathway is known to produce reactive oxidative species. We observed an upregulation of genes involved in the protection against reactive oxidative species. Key genes involved in de novo lipogenesis and lipid transport were also upregulated. Finally, genes involved in DNA damage control and unfolded protein responses were downregulated.

What the primary care pediatrician should know about syndromes associated with exposures to mycotoxins.

Disease associated with exposure to mycotoxins is known as the "Great Masquerader" of the 21st century because of its complex natural history involving different tissues and resembling different diseases at each stage in its evolution. It can present with a variety of nonspecific clinical signs and symptoms such as rash, conjunctivitis, epistaxis, apnea, cough, wheezing, nausea, and vomiting. Some cases of vomiting illness, bone marrow failure, acute pulmonary hemorrhage, and recurrent apnea and/or "pneumonia" are associated with exposure to mycotoxins. Familiarity with the symptoms of exposure to the major classes of mycotoxins enables the clinician to ask pertinent questions about possible fungal exposures and to remove the infant or child from the source of exposure, which could be contaminated food(s), clothing and furniture, or the indoor air of the home. Failure to prevent recurrent exposure often results in recurrent illness. A variety of other conditions, including hepatocellular and esophageal cancer and neural tube defects, are associated with consumption of foods contaminated with mycotoxins. Awareness of the short- and long-term consequences of exposures to these natural toxins helps pediatricians to serve as better advocates for children and families.

Aflatoxicosis, infectious bursal disease and immune response to Newcastle disease vaccination in rural chickens.

To investigate the immunosuppressive effects of infectious bursal disease virus (IBDV) and aflatoxin in indigenous chickens of Uganda, Newcastle disease (ND) seronegative chicks were randomly allocated to two treatment groups. Group A chicks were injected intramuscularly at the age of 3 weeks every 2 days up to four times with 0.250 mg aflatoxin B1 per bird, group B was infected occulo-nasally with IBDV 3 days prior to vaccination, while group C was left as a control group. All the chicks from the three groups were then vaccinated with Hitchner B1 vaccine at 21 days of age followed by a secondary vaccination with La Sota vaccine 3 weeks later. Humoral and cell-mediated immune responses were assessed by measuring antibody levels and delayed hypersensitivity reaction post vaccination. Growth performance in the three groups was assessed by weekly body weights while evidence of excretion of vaccinal ND virus was detected by reverse transcription-polymerase chain reaction.A significant (P < 0.05) reduction in the haemagglutination inhibition of ND antibody titre following initial priming with Hitchner B1 and subsequent booster with La Sota vaccines and a delayed hypersensitivity test following sensitization with dinitrochlorobenzene showed aflatoxin to be a more potent immunosuppressant than IBDV. Aflatoxin exerted its maximum effects during primary antibody response in the second and third weeks post vaccination. Aflatoxin and IBDV did not affect growth rates (P > 0.05) but prolonged La Sota vaccine virus excretion in faeces. Under our experimental conditions, aflatoxin and IBDV do not significantly affect the immune response of rural chickens to ND vaccination.

Preliminary results of monitoring research on zearalenone presence in blood of women with neoplastic lesions in reproductive system.

The aim of the monitoring of zearalenone presence in the blood plasma of women with neoplastic lesions in the reproductive tract was to asses whether the phytosteride is noted in the patients blood and whether the correlation exists between its presence and the incidence of particular neoplasm. The presence of zearalenone or its metabolite--alpha-zearalenole, was noted in 13.51% of the examined women. In 60% of the patients with the confirmed presence of the xenobiotic it was noted in the lowest concentrations. These patients had neoplastic lesions of Carcinoma corpus uteri type.

Clinical implications of mycotoxins and Stachybotrys.

There has been considerable interest and concern in recent years regarding the potential health effects of mycotoxins in the indoor environment. Although the existence of mycotoxins has been known for several decades, relatively little is known about their effects in humans. What is known comes almost exclusively from studies of ingestion as the route of exposure. This review summarizes what is known regarding health effects of mycotoxins in general and specifically examines the evidence for the role of indoor exposure to the fungi of the genus Stachybotrys as a cause of disease in humans. Much work remains to be done in the area of mycotoxin research. The risk of health effects from ingestion seems much more widespread than from indoor airborne exposure, although the latter has received considerably more media attention. Rigorously controlled studies are needed to clarify these issues.

Tremorgenic mycotoxicosis in four dogs from a single household.

Mycotoxins are fungal metabolites that induce undesirable effects. The effects of these mycotoxins vary depending on the chemical structure of the toxin and degree of toxicity. Mycotoxins that induce muscle tremors, ataxia, and convulsions are termed tremorgenic mycotoxins. Our report documents the clinical course of 4 dogs from a single household that were simultaneously affected by tremorgenic mycotoxins. Diagnosis of tremorgenic mycotoxicosis was confirmed by stomach content analysis from 1 of the dogs. The mycotoxins identified were penitrem A and roquefortine, which are both produced by Penicillium spp. Treatment goals following tremorgenic mycotoxin ingestion include minimizing absorption, controlling tremors and seizures with methocarbamol and pentobarbital sodium administration, and providing supportive care. Two of the affected dogs required ventilatory support. With early aggressive treatment, prognosis is good and recovery is complete without sequelae. It is helpful for the clinician to be familiar with the typical clinical signs at the time of admission, treatment, and clinical course of dogs with tremorgenic mycotoxicosis.

Retos en el diagnostico de las intoxicaciones por setas

La recolección y consumo de setas es una afición cada vez más popular en nuestro país. Los micetismos constituyen una variedad de síndromes clínicos bien descritos si bien se siguen identificando especies tóxicas o nuevas patologías. Esto queda ejemplificado en la asociación reciente de un cuadro de rabdomiolisis después de haber ingerido Tricholoma equestre, o seta de los caballeros, considerada desde antiguo como un comestible de gran calidad. Por otro lado en el área de la Toxinología se precisa aplicar una medicina basada en la evidencia siendo básico el conocimiento de la epidemiología de los micetismos en cada centro médico. El análisis de laboratorio sirve para confirmar el diagnóstico de un micetismo, evitar tratamientos y hospitalizaciones innecesarias, diagnosticar síndromes mixtos o micetismos cuyo periodo de incubación quede solapado y aportar nuevos taxones al catálogo de especies tóxicas. Las muestras disponibles van a condicionar el análisis a realizar. Con el estudio de restos botánicos se llega a establecer al menos el género en más del 80 por ciento de los casos. La búsqueda de las toxinas en muestras botánicas, y especialmente de amanitinas en orina, permite el diagnóstico confirmativo. La dificultad que presenta el estudio morfológico y la determinación de toxinas en muestras en mal estado de conservación, hace que la aplicación de marcadores genéticos pudiera ser una buena herramienta complementaria. La creación de una base de datos de las regiones con interés en la identificación genética de setas tóxicas de la península ibérica, está siendo objeto de investigación en nuestro laboratorio (AU)

Studies of sick building syndrome. IV. Mycotoxicosis.

There has been increasing public attention to the potential health risks of mold exposure, particularly in wet buildings. A variety of molds has been isolated from both damaged homes and businesses, including agents that secrete toxigenic materials. One area that is attracting particular notice is the relative toxigenic potential of mycotoxins. Although exposure to molds can produce significant mucosal irritation, there are very few data to suggest long-term ill effects. More importantly, there is no evidence in humans that mold exposure leads to nonmucosal pathology. In fact, many of the data on toxigenic molds are derived from animal toxicity studies, and these are based primarily, on ingestion. Although every attempt should be made to improve the quality of indoor air, including avoidance of molds, the human illnesses attributed to fungal exposure are, with the exception of invasive infections and mold allergy, relatively rare. In this review we discuss selected aspects of the microbiology of mycotoxin-producing molds and their potential role in human immunopathology with respect to wet building environments.

Influence of protein supplementation and implant status on alleviating fescue toxicosis.

Heat stress is a major problem in transporting stocker calves with symptoms of fescue toxicosis. Removing calves from tall fescue pastures and offering diets devoid of endophyte-infected tall fescue could reduce the severity of toxicosis and precondition calves for transport to the feedlot. In the present experiment, a pasture phase was used to condition yearling steers to grazing tall fescue and induce symptoms of fescue toxicosis, and a pen phase followed to determine effects of implanting at the start of grazing and protein supplementation (hay only vs hay plus supplement) on short-term changes in rectal temperature and serum prolactin concentration. Neither implant status nor protein supplementation affected (P > 0.10) white blood cell count or rectal temperature. White blood cell counts at the conclusion of the pasture phase averaged 8,778 cells/microL and were within a range indicating no immunological response. Changes in rectal temperature and serum prolactin concentration during the pen phase were not influenced (P > 0.10) by implanting or supplementation. Initial rectal temperatures for the pen phase were high (39.9 degrees C) but declined linearly (P < 0.001) over the first 106 h and were below a normal temperature (39.2 degrees C) by 82 h following removal from tall fescue pastures. Serum prolactin gradually increased (P < 0.001) to a peak by 82 h and stabilized thereafter. Results indicate that neither supplemental protein nor an estrogenic implant influenced recovery indices of fescue toxicosis, whereas removing calves from tall fescue pastures and excluding dietary tall fescue for 3 to 4 d may alleviate symptoms of fescue toxicosis.

Ameliorative effects of sodium bentonite on phagocytosis and Newcastle disease antibody formation in broiler chickens during aflatoxicosis.

The ameliorative effect of graded levels of dietary sodium bentonite (0.2, 0.4 and 0.6 per cent wt/wt of feed) on in vitro-impaired phagocytosis and suppressed immune response to Newcastle disease vaccine during aflatoxicosis (AF) in broiler chicks was investigated. Both percentage and mean of phagocytic activities were decreased significantly (P < 0.05) in chicks fed 2.5 mg aflatoxin per kg feed. The addition of sodium bentonite was significantly effective in ameliorating the negative effect of AF on the percentage and mean of phagocytosis. The presence of AF alone in the diet depressed the immune response of chicks as measured by haemagglutination inhibition (HI) test. Sodium bentonite was also effective in ameliorating the suppressive effect of AF on the HI -titre in chicks vaccinated against Newcastle disease. The best results obtained when sodium bentonite was added at the rate of 0.4 per cent wt/wt of feed to the AF-containing diets.

Influence of dietary aflatoxin on the severity of coccidial infection in quails.

One hundred-sixty one day old Japanese quail-chicks were divided into four groups and kept for 6 weeks. The first group was kept as control. Group 2 received 1 ppm of dietary aflatoxin from day zero of chick life till the end of experiment. Group 3 was inoculated with 5 x 10(4) sporulated oocysts of Eimeria alone. Quail-chicks of group 4 were inoculated with coccidial oocysts orally and received aflatoxin in their rations. The combination of aflatoxin and coccidia produced higher mortality rate, higher faecal scores with increased oocysts output and reproductive potential than those quails received aflatoxin or coccidia alone. Body weight and efficiency of feed utilization decreased in all treated groups. The maximal losses of body gain and efficiency of feed utilization were recorded in quail-chicks infected with Eimeria and received aflatoxin in their ration. The lowest erythrocytic count, hemoglobin values, PCV and leukocytic count were recorded in quails that infected with coccidia and received aflatoxin. The levels of total serum proteins, gamma globulins, calcium and phosphorus were decreased in case of quails infected with coccidia and received dietary aflatoxin. Total bilirubin and GOT activity appeared to be higher in quails infected with Eimeria and received aflatoxin.

Black soil blindness: a new mycotoxicosis of cattle grazing Corallocytostroma-infected Mitchell grass (Astrebla spp).

A new, fatal mycotoxicosis of cattle has been recognised in north-western Australia. A feeding trial confirmed the toxicity of a previously unknown species of Corallocytostroma that grows on Mitchell grass (Astrebla spp). The disease has been colloquially named 'black soil blindness' because its most prominent features are its confinement to pastures on black soil, and blindness and death of affected animals. Over 500 cattle have died and considerable subclinical disease in present. Above average wet season rainfall and extended growing seasons may explain the emergence of the fungus. The disease is important because cattle production in large areas of Australia utilise Mitchell grass pastures.

An outbreak of acute hepatic encephalopathy due to severe aflatoxicosis in Malaysia.

In October 1988, 13 Chinese children died of acute hepatic encephalopathy in the northwestern state of Perak in peninsular Malaysia. The acuteness of the illness differed from previously reported outbreaks described in Kenya, India, and Thailand. Epidemiologic investigations determined that the children had eaten a Chinese noodle, loh see fun, hours before they died. The attack rates among those who had eaten the noodles were significantly higher than those who had not (P < 0.0001). The cases were geographically scattered in six towns in two districts along the route of distribution of the noodle supplied by one factory in Kampar town. Aflatoxins were confirmed in postmortem samples from patients. This outbreak has important public health implications for many developing countries.