Aflatoxinas: conceitos sobre mecanismos de toxicidade e seu envolvimento na etiologia do câncer hepático celular

Foram revistos os conceitos de maior relevância sobre mecanismos de toxicidade e evidências do envolvimento das aflatoxinas na etiologia do câncer hepático humano. A aflatoxina B1 (AFB1), principal metabólito produzido por fungos do gênero Aspergillus, manifesta seus efeitos tóxicos após conversão hepática em AFB1-epóxido, o qual reage com macromoléculas celulares, incluindo proteínas, RNA (ácido ribonucléico) e DNA (ácido desoxirribonucléico). A reação com o DNA ocorre através da ligação com guaninas, ao nível do códon 249, do gene supressor de tumores p53. Em seres humanos, estudos de biomonitoramento individual de derivados AFB1-N7-guanina tem demonstrado que as aflatoxinas constituem importantes fatores de risco, com uma provável interação sinergística com o vírus da hepatite B, para o desenvolvimento do carcinoma hepatocelular em populações expostas. Considerando-se a ocorrência freqüente das aflatoxinas em produtos alimentícios, no Brasil, ressalta-se a necessidade de estudos que avaliem criteriosamente o impacto dos níveis de exposição a estas toxinas sobre a saúde humana.

Air contaminants in animal production: the poultry case

A descriptive study was developed in order to assess air contamination caused by fungi and particles in seven poultry units. Twenty seven air samples of 25 litters were collected through impaction method. Air sampling and particle concentration measurement were performed in the pavilions’ interior and also outside premises, since this was the place regarded as reference. Simultaneously, temperature and relative humidity were also registered. Regarding fungal load in the air from the seven poultry farms, the highest value obtained was 24040 CFU/m3 and the lowest was 320 CFU/m3. Twenty eight species/genera of fungi were identified, being Scopulariopsis brevicaulis (39.0%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. From the Aspergillus genus, Aspergillus flavus (74.5%) was the most frequently detected species. There was a significant correlation (r=0.487; p=0.014) between temperature and the level of fungal contamination (CFU/m3). Considering contamination caused by particles, in this study, particles with larger dimensions (PM5.0 and PM10) have higher concentrations. There was also a significant correlation between relative humidity and concentration of smaller particles namely, PM0.5 (r=0.438; p=0.025) and PM1.0 (r=0.537; p=0.005). Characterizing typical exposure levels to these contaminants in this specific occupational setting is required to allow a more detailed risk assessment analysis and to set exposure limits to protect workers’ health.

Avaliação da qualidade química e microbiológica de tintas didácticas do ensino pré-escolar

As tintas utilizadas nas actividades didácticas possuem na sua composição ingredientes que dada a sua natureza, modo de fabrico e de utilização, podem representar um risco para a saúde das crianças. Neste âmbito, procurou-se com este estudo avaliar a qualidade química e microbiológica das tintas utilizadas pelas crianças no ensino pré-escolar. Vinte e nove amostras de tintas, incluindo guaches, tintas de águas, digitintas e tintas para pinturas faciais foram recolhidas em oito estabelecimentos de ensino, nomeadamente, Jardins de Infância, do conselho de Vila Nova de Gaia. A avaliação microbiológica envolveu não só a determinação da concentração microbiana presente nas amostras, como também, a avaliação da estabilidade microbiana nas tintas das espécies S.aureus e E.coli. Na avaliação química procedeu-se à determinação da concentração dos metais chumbo (Pb), cádmio (Cd), crómio (Cr), cobalto (Co), níquel (Nq), manganês (Mn), cobre (Cu) e zinco (Zn) quer em algumas das amostras recolhidas nos estabelecimentos de ensino, quer em tintas adquiridas em três estabelecimentos comerciais. Os resultados obtidos da avaliação microbiológica revelam uma contaminação estática na generalidade das tintas. Três amostras de tintas apresentaram ainda elevada contaminação por fungos, nomeadamente Aspergillus spp. e Trichophyton spp. Da avaliação da estabilidade microbiana das espécies S.aureus e E.coli observou-se uma sensibilidade das mesmas às tintas, sendo evidenciado, em alguns casos, um decrescimento da concentração ao longo do tempo de exposição, e noutros, uma sensibilidade imediata. A espécie S.aureus revelou, contudo, maior capacidade de resistência que a E.coli. Os resultados obtidos da avaliação química revelaram a presença de Cr em todas as amostras, registando as tintas adquiridas em estabelecimentos comerciais concentrações mais elevadas para este metal. Os metais Cu e Zn foram detectados, em algumas amostras de tintas artísticas, em concentrações acima dos valores limites. Nas tintas para a cara foram encontrados os metais Pb, Cd, Cr e Nq, cuja utilização é interdita nestes produtos. O conhecimento das características químicas e microbiológicas das tintas utilizadas por crianças do ensino pré-escolar revelou-se de grande importância, nomeadamente, para a determinação dos riscos a que este grupo de indivíduos pode estar exposto no seu dia-a-dia quando utilizam estes produtos.

Fungal and microbial volatile organic compounds exposure assessment in a waste sorting plant

In the management of solid waste, pollutants over a wide range are released with different routes of exposure for workers. The potential for synergism among the pollutants raises concerns about potential adverse health effects, and there are still many uncertainties involved in exposure assessment. In this study, conventional (culture-based) and molecular real-time polymerase chain reaction (RTPCR) methodologies were used to assess fungal air contamination in a waste-sorting plant which focused on the presence of three potential pathogenic/toxigenic fungal species: Aspergillus flavus, A. fumigatus, and Stachybotrys chartarum. In addition, microbial volatile organic compounds (MVOC) were measured by photoionization detection. For all analysis, samplings were performed at five different workstations inside the facilities and also outdoors as a reference. Penicillium sp. were the most common species found at all plant locations. Pathogenic/toxigenic species (A. fumigatus and S. chartarum) were detected at two different workstations by RTPCR but not by culture-based techniques. MVOC concentration indoors ranged between 0 and 8.9 ppm (average 5.3 ± 3.16 ppm). Our results illustrated the advantage of combining both conventional and molecular methodologies in fungal exposure assessment. Together with MVOC analyses in indoor air, data obtained allow for a more precise evaluation of potential health risks associated with bioaerosol exposure. Consequently, with this knowledge, strategies may be developed for effective protection of the workers.

Fungal contamination of poultry litter: a public health problem

Although numerous studies have been conducted on microbial contaminants associated with various stages related to poultry and meat products processing, only a few reported on fungal contamination of poultry litter. The goals of this study were to (1) characterize litter fungal contamination and (2) report the incidence of keratinophilic and toxigenic fungi presence. Seven fresh and 14 aged litter samples were collected from 7 poultry farms. In addition, 27 air samples of 25 litters were also collected through impaction method, and after laboratory processing and incubation of collected samples, quantitative colony-forming units (CFU/m3) and qualitative results were obtained. Twelve different fungal species were detected in fresh litter and Penicillium was the most frequent genus found (59.9%), followed by Alternaria (17.8%), Cladosporium (7.1%), and Aspergillus (5.7%). With respect to aged litter, 19 different fungal species were detected, with Penicillium sp. the most frequently isolated (42.3%), followed by Scopulariopsis sp. (38.3%), Trichosporon sp. (8.8%), and Aspergillus sp. (5.5%). A significant positive correlation was found between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m3). Litter fungal quantification and species identification have important implications in the evaluation of potential adverse health risks to exposed workers and animals. Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genus) as well as toxigenic fungi (Aspergillus, Fusarium, and Penicillium genus) were isolated.

Occupational exposure to aflatoxin (AFB1) in poultry production

Aflatoxin B1 (AFB1) has been recognized to produce cancer in human liver. In addition, epidemiological and laboratory studies demonstrated that the respiratory system was a target for AFB1. Exposure occurs predominantly through the food chain, but inhalation represents an additional route of exposure. The present study aimed to examine AFB1 exposure among poultry workers in Portugal. Blood samples were collected from a total of 31 poultry workers from six poultry farms. In addition, a control group (n = 30) was included comprised of workers who undertook administrative tasks. Measurement of AFB1 in serum was performed by enzyme-linked immunosorbent assay (ELISA). For examining fungi contamination, air samples were collected through an impaction method. Air sampling was obtained in pavilion interior and outside the premises, since this was the place regarded as the reference location. Using molecular methods, toxicogenic strains (aflatoxin-producing) were investigated within the group of species belonging to Aspergillus flavus complex. Eighteen poultry workers (59%) had detectable levels of AFB1 with values ranging from <1 ng/ml to4.23 ng/ml and with a mean value of 2 ± 0.98ng/ml. AFB1 was not detected in the serum sampled from any of the controls. Aspergillus flavus was the fungal species third most frequently found in the indoor air samples analyzed (7.2%) and was the most frequently isolated species in air samples containing only Aspergillus genus (74.5%). The presence of aflatoxigenic strains was only confirmed in outdoor air samples from one of the units, indicating the presence of a source inside the building in at least one case. Data indicate that AFB1 inhalation represents an additional risk in this occupational setting that needs to be recognized, assessed, and prevented.

Fungal contamination in swine: a potential occupational health threat

Poor air quality in a pig-confinement building may potentially place farmers at higher health risk than other workers for exposure to airborne pollutants that may reach infectious levels. The aim of this study was to assess worker exposure to fungi in indoor environments in Portuguese swine buildings. Air samples from 7 swine farms were collected at a flow rate of 140 L/min, at 1 m height, onto malt extract agar supplemented with chloramphenicol (MEA). Surfaces samples of the same indoor sites were obtained by swabbing the surfaces. Samples from the floor covering were also collected from four of seven swine farms. All collected samples were incubated at 27°C for 5-7 days. After lab processing and incubation of obtained samples, quantitative colony-forming units (CFU)/m(3), CFU/cm(2), and CFU/g and qualitative results were determined with identification of isolated fungal species. Aspergillus versicolor was the most frequent species found in air (21%), followed by Scopulariopsis brevicaulis (17%) and Penicillium sp. (14%). Aspergillus versicolor was also the most frequent species noted on surfaces (26.6%), followed by Cladosporium sp. (22.4%) and Scopulariopsis brevicaulis (17.5%). Chrysosporium was the most frequently found genera in the new floor covering (38.5%), while Mucor was the most prevalent genera (25.1%) in used floor covering. Our findings corroborate a potential occupational health threat due to fungi exposure and suggest the need for a preventive strategy.

Monitoramento de fungos anemófilos e de leveduras em unidade hospitalar

OBJETIVO: Monitorar e caracterizar fungos anemófilos e leveduras de fontes bióticas e abióticas de uma unidade hospitalar. MÉTODOS: As coletas foram realizadas mensalmente e em dois períodos, do centro cirúrgico e unidades de terapia intensiva adulto e neonatal em hospital de Araraquara, Estado de São Paulo. Para coleta de fungos anemófilos foi utilizado amostrador tipo Andersen de simples estágio. A pesquisa de leveduras foi feita das mãos e de orofaringe de profissionais de saúde, bem como de superfícies de leitos e de maçanetas das áreas críticas. RESULTADOS: Foram recuperados do centro cirúrgico 32 gêneros de fungos anemófilos e 31 das unidades de terapia intensiva. Os gêneros mais freqüentemente isolados foram Cladophialophora spp., Fusarium spp., Penicillium spp., Chrysosporium spp. e Aspergillus spp. Durante o período de estudo, houve reforma e implantação de uma unidade dentro do hospital, que coincidiu com o aumento na contagem de colônias de Cladophialophora spp., Aspergillus spp. e Fusarium spp. Leveduras foram encontradas em 39,4% dos profissionais de saúde (16,7% das amostras dos espaços interdigitais, 12,1% do leito subungueal e 10,6% da orofaringe) e, em 44% das amostras do mobiliário, com predomínio do gênero Candida (C. albicans, C. guilliermondii, C. parapsilosis e C. lusitaniae) seguido por Trichosporon spp. CONCLUSÕES: Observou-se número relativamente elevado de fungos anemófilos (potencialmente patogênicos) em áreas especiais e níveis expressivos de leveduras em fontes bióticas e abióticas. O monitoramento microbiológico ambiental deve ser realizado, principalmente em salas especiais com pacientes imunocomprometidos, sujeitos à exposição de patógenos do meio ambiente, assim como, advindos de profissionais de saúde.

Polycyclic aromatic hydrocarbon levels in three pelagic fish species from Atlantic Ocean: inter-specific and inter-season comparisons and assessment of potential public health risks

The concentrations of 18 polycyclic aromatic hydrocarbons (PAHs) were determined in three commercially valuable fish species (sardine, Sardina pilchardus; chub mackerel, Scomber japonicus; and horse mackerel, Trachurus trachurus) from the Atlantic Ocean. Specimens were collected seasonally during 2007–2009. Only low molecular weight PAHs were detected, namely, naphthalene, acenaphthene, fluorene and phenanthrene. Chub mackerel (1.80–19.90 microg/kg ww) revealed to be significantly more contaminated than horse mackerel (2.73–10.0 microg/kg ww) and sardine (2.29–14.18 microg/kg ww). Inter-specific and inter-season comparisons of PAHs bioaccumulation were statistically assessed. The more relevant statistical correlations were observed between PAH amounts and total fat content (significant positive relationships, p < 0.05), and season (sardine displayed higher amounts in autumn–winter while the mackerel species showed globally the inverse behavior). The health risks by consumption of these species were assessed and shown to present no threat to public health concerning PAH intakes.

Lipid content of frozen fish: comparison of different extraction methods and variability during freezing storage

In this work, a microwave-assisted extraction (MAE) methodology was compared with several conventional extraction methods (Soxhlet, Bligh & Dyer, modified Bligh & Dyer, Folch, modified Folch, Hara & Radin, Roese-Gottlieb) for quantification of total lipid content of three fish species: horse mackerel (Trachurus trachurus), chub mackerel (Scomber japonicus), and sardine (Sardina pilchardus). The influence of species, extraction method and frozen storage time (varying from fresh to 9 months of freezing) on total lipid content was analysed in detail. The efficiencies of methods MAE, Bligh & Dyer, Folch, modified Folch and Hara & Radin were the highest and although they were not statistically different, differences existed in terms of variability, with MAE showing the highest repeatability (CV = 0.034). Roese-Gottlieb, Soxhlet, and modified Bligh & Dyer methods were very poor in terms of efficiency as well as repeatability (CV between 0.13 and 0.18).

Occupational exposure to Aflatoxin B1 in swine production and possible contamination sources

Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n  = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.

Exposure to particles and fungi in Portuguese swine production

Versão preprint.

Accessing indoor fungal contamination using conventional and molecular methods in Portuguese poultries

Epidemiological studies showed increased prevalence of respiratory symptoms and adverse changes in pulmonary function parameters in poultry workers, corroborating the increased exposure to risk factors, such as fungal load and their metabolites. This study aimed to determine the occupational exposure threat due to fungal contamination caused by the toxigenic isolates belonging to the complex of the species of Aspergillus flavus and also isolates fromAspergillus fumigatus species complex. The study was carried out in seven Portuguese poultries, using cultural and molecularmethodologies. For conventional/cultural methods, air, surfaces, and litter samples were collected by impaction method using the Millipore Air Sampler. For the molecular analysis, air samples were collected by impinger method using the Coriolis μ air sampler. After DNA extraction, samples were analyzed by real-time PCR using specific primers and probes for toxigenic strains of the Aspergillus flavus complex and for detection of isolates from Aspergillus fumigatus complex. Through conventional methods, and among the Aspergillus genus, different prevalences were detected regarding the presence of Aspergillus flavus and Aspergillus fumigatus species complexes, namely: 74.5 versus 1.0% in the air samples, 24.0 versus 16.0% in the surfaces, 0 versus 32.6% in new litter, and 9.9 versus 15.9%in used litter. Through molecular biology, we were able to detect the presence of aflatoxigenic strains in pavilions in which Aspergillus flavus did not grow in culture. Aspergillus fumigatus was only found in one indoor air sample by conventional methods. Using molecular methodologies, however, Aspergillus fumigatus complex was detected in seven indoor samples from three different poultry units. The characterization of fungal contamination caused by Aspergillus flavus and Aspergillus fumigatus raises the concern of occupational threat not only due to the detected fungal load but also because of the toxigenic potential of these species.

Mould and yeast identification in archival settings: preliminary results on the use of traditional methods and molecular biology options in Portuguese archives

This project was developed to fully assess the indoor air quality in archives and libraries from a fungal flora point of view. It uses classical methodologies such as traditional culture media – for the viable fungi – and modern molecular biology protocols, especially relevant to assess the non-viable fraction of the biological contaminants. Denaturing high-performance liquid chromatography (DHPLC) has emerged as an alternative to denaturing gradient gel electrophoresis (DGGE) and has already been applied to the study of a few bacterial communities. We propose the application of DHPLC to the study of fungal colonization on paper-based archive materials. This technology allows for the identification of each component of a mixture of fungi based on their genetic variation. In a highly complex mixture of microbial DNA this method can be used simply to study the population dynamics, and it also allows for sample fraction collection, which can, in many cases, be immediately sequenced, circumventing the need for cloning. Some examples of the methodological application are shown. Also applied is fragment length analysis for the study of mixed Candida samples. Both of these methods can later be applied in various fields, such as clinical and sand sample analysis. So far, the environmental analyses have been extremely useful to determine potentially pathogenic/toxinogenic fungi such as Stachybotrys sp., Aspergillus niger, Aspergillus fumigatus, and Fusarium sp. This work will hopefully lead to more accurate evaluation of environmental conditions for both human health and the preservation of documents.

Occupational exposure to particulate matter and fungi in a composting plant: case study in Portugal

The handling of waste can be responsible for occupational exposure to particles and fungi. The aim of this study was to characterize exposure to particles and fungi in a composting plant. Measurements of particulate matter were performed using portable direct-reading equipment. Air samples of 50L were collected through an impaction method with a flow rate of 140L/min onto malt extract agar supplemented with chloramphenicol (0.05%). Surfaces samples were also collected. All the samples were incubated at 27ºC for 5 to 7 days. Particulate matter data showed higher contamination for PM, and PM10 sizes. Aspergillus genus presents the highest air prevalence (90.6%). Aspergillus niger (32.6%), A. fumigatus (26.5%) and A. flavus (16.3%) were the most prevalent fungi in air sampling, and Mucor sp. (39.2%), Aspergillus niger (30.9%) and A. fumigatus (28.7%) were the most found in surfaces. the results obtained claim the attention to the need of further research.