RESUMEN
The increasing spread of ragworts is observed with concern. Ragworts like tansy ragwort (Jacobaea vulgaris Gaertn.) or marsh ragwort (J. aquatica) contain pyrrolizidine alkaloids (PA) which may induce hepatotoxic effects. Grazing animals usually avoid ragworts if their pasture management is appropriate. Preserved feed prepared from ragworts contaminated meadows may, however, lead to a significant exposure to PA. Previous studies on toxicity of PA for dairy cows revealed inconsistent results due to feeding ragwort plant material which was associated with heterogeneous PA exposure and thus failed to conclusively deduce critical PA doses. Therefore, the aim of the present study was to expose dairy cows (n = 4 per group) in a short-term scenario for 28 days with increasing PA doses (PA1: 0.47 mg PA/kg body weight (BW)/day (d); PA2: 0.95 mg PA/kg BW/d; PA3: 1.91 mg PA/kg BW/d) via oral administration by gavage of a defined PA-extract. While group PA3 was dosed with the PA-extract alone, groups PA2 and PA1 received PA-extracts blended in similar volumes with molasses to provide comparable amounts of sugar. Additionally, two control groups were treated either with water (CONWater) or with molasses (CONMolasses) to assess the effects of sugar without PA interference. While clinical traits including dry matter intake, milking performance, rectal body temperature, ruminal activity and body condition score (BCS) were not influenced by PA exposure, activities of enzymes indicative for liver damages, such as gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH), increased significantly over time at an exposure of 1.91 mg total PA/kg BW/d.
Asunto(s)
Alcaloides de Pirrolicidina , Senecio , Tanacetum , Femenino , Bovinos , Animales , Alcaloides de Pirrolicidina/toxicidad , Dieta/veterinaria , Alimentación Animal/análisis , Agua , Extractos Vegetales , AzúcaresRESUMEN
1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid-liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.
Asunto(s)
Alcaloides de Pirrolicidina , Animales , Cromatografía Liquida/métodos , Alcaloides de Pirrolicidina/análisis , Leche/química , Espectrometría de Masas en Tándem/métodos , Tropanos/análisis , Cromatografía Líquida de Alta PresiónRESUMEN
Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.
Asunto(s)
Alcaloides de Pirrolicidina , Animales , Carcinógenos/farmacología , Humanos , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Alcaloides de Pirrolicidina/química , ToxicocinéticaRESUMEN
Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay's detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.
Asunto(s)
Genotipo , Compuestos Macrocíclicos/metabolismo , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Stachybotrys/genética , Stachybotrys/metabolismo , Tricotecenos/metabolismo , Compuestos Macrocíclicos/química , Sensibilidad y Especificidad , Tricotecenos/químicaRESUMEN
1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.
Asunto(s)
Hepatocitos/metabolismo , Alcaloides de Pirrolicidina/metabolismo , Activación Metabólica , Transporte Biológico Activo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Humanos , Estructura Molecular , Alcaloides de Pirrolicidina/química , Alcaloides de Pirrolicidina/farmacología , Alcaloides de Pirrolicidina/toxicidadRESUMEN
Straw is the main by-product of grain production, used as bedding material and animal feed. If produced or stored under adverse hygienic conditions, straw is prone to the growth of filamentous fungi. Some of them, e.g. Aspergillus, Fusarium and Stachybotrys spp. are well-known mycotoxin producers. Since studies on mycotoxins in straw are scarce, 192 straw samples (wheat n = 80; barley n = 79; triticale n = 12; oat n = 11; rye n = 12) were collected across Germany within the German official feed surveillance and screened for the presence of 21 mycotoxins. The following mycotoxins (positive samples for at least one mycotoxin n = 184) were detected: zearalenone (n = 86, 6.0-785 µg/kg), nivalenol (n = 51, 30-2,600 µg/kg), deoxynivalenol (n = 156, 20-24,000 µg/kg), 15-acetyl-deoxynivalenol (n = 34, 20-2,400 µg/kg), 3-acetyl-deoxynivalenol (n = 16, 40-340 µg/kg), scirpentriol (n = 14, 40-680 µg/kg), T-2 toxin (n = 67, 10-250 µg/kg), HT-2 toxin (n = 92, 20-800 µg/kg), T-2 tetraol (n = 13, 70-480 µg/kg). 15-monoacetoxyscirpenol (30 µg/kg) and T-2 triol (60 µg/kg) were only detected in one barley sample. Macrocyclic trichothecenes (satratoxin G, F, roridin E, and verrucarin J) were also found in only one barley sample (quantified as roridin A equivalent: total 183 µg/kg). The occurrence of stachybotrylactam was monitored for the first time in four samples (n = 4, 0.96-7.4 µg/kg). Fusarenon-X, 4,15-diacetoxyscirpenol, neosolaniol, satratoxin H and roridin-L2 were not detectable in the samples. The results indicate a non-negligible contribution of straw to oral and possibly inhalation exposure to mycotoxins of animals or humans handling contaminated straw.
Asunto(s)
Alimentación Animal/análisis , Ensilaje/análisis , Tricotecenos/análisis , Zearalenona/análisis , Dieta/veterinaria , AlemaniaRESUMEN
Stachybotrys (S.) chartarum is a cellulolytic mould with the ability to produce highly cytotoxic macrocyclic trichothecenes. Two chemotypes are defined according to their ability to produce either atranones or satratoxins. S. chartarum has been well known as the causative agent of the lethal disease stachybotryotoxicosis in horses. Further investigations revealed that this disease is strictly correlated with the presence of macrocyclic trichothecenes. Furthermore, their occurrence in water-damaged buildings has been linked to adverse health effects such as the sick building syndrome. As the chemotypes cannot be characterized via phenotypic criteria, different methods such as PCR, MALDI-TOF MS, LC-MS/MS, thin-layer chromatography and cytotoxicity assays have been used so far. Fourier-transform-infrared spectroscopy (FT-IR) is commonly used for the differentiation of bacteria and yeasts, but this technique is also applicable to filamentous fungi. Hence, this study aimed at evaluating to which extent a reliable differentiation of S. chartarum chemotypes A and S is possible. Besides, another objective was to verify if the recently introduced third genotype of S. chartarum can be identified. Therefore, 28 strains including the two chemotypes and the third genotype H were cultivated on malt extract agar (MEA) and potato dextrose agar in three biological replicates. Each sample was applied to FT-IR measurements on day 7, 14 and 21 of cultivation. In this study, we achieved a distinction of the chemotypes A and S via FT-IR spectroscopy after incubation for 7 days on MEA. In terms of genotype differentiation, the PCR detecting satratoxin- and atranone-gene clusters remained the only applicable method.
Asunto(s)
Espectroscopía Infrarroja por Transformada de Fourier , Stachybotrys , Animales , Genotipo , Caballos , Stachybotrys/clasificaciónRESUMEN
Pyrrolizidine alkaloids (PA) and PA-N-oxides (PANO) are a large group of secondary plant metabolites comprising more than 660 compounds. Exhibiting geno- and hepatotoxic properties, they are responsible for multiple cases of food and feed poisoning over the last 100 years. For food and feed safety reasons, relevant PA/PANO should be monitored extensively in the main sources of PA/PANO intake. In this study, a sensitive analytical method was developed for detecting a broad range of 44 commercially available PA/PANO compounds, and in-house validation procedures were performed for several (herbal) teas. Various extraction solvents and procedures, as well as solid phase extraction materials for sample clean-up and analyte concentration, were tested to establish the methods' efficiency and effectiveness. Chromatographic conditions were optimised to obtain the best possible separation of isomers for the 44 PA/PANO analytes. The final method was proven very sensitive and accurate, with detection limits ranging from 0.1 to 7.0 µg/kg and precisions between 0.7 and 16.1%. For 40 of the analytes, the recovery rates ranged from 60.7 to 128.8%. The applicability and trueness of the method were examined by analysing tea samples from a local supermarket and comparing them to a reference material. At least one PA/PANO analyte was detected in 17 of the 18 samples under investigation, and the sum contents of the samples ranged from 0.1 to 47.9 µg/kg. Knowledge of the PA/PANO composition in a sample can be used to indicate the botanical origin of the impurity and, thus, the geographical region of cultivation.
Asunto(s)
Cromatografía Liquida/métodos , Alcaloides de Pirrolicidina/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Té/química , Tés de Hierbas/análisis , Contaminación de Alimentos/análisis , Límite de Detección , Reproducibilidad de los ResultadosRESUMEN
The Bacillus (B.) cereus group consists of nine recognized species which are present worldwide. B. cereus play an important role in food-borne diseases by producing different toxins. Yet, only a small percentage of B. cereus strains are able to produce the heat stable cereulide, the causative agent of emetic food poisoning. To minimize the entry of emetic B. cereus into the food chain, food business operators are dependent on efficient and reliable methods enabling the differentiation between emetic and non-emetic strains. Currently, only time-consuming cell bioassays, molecular methods and tandem mass spectrometry are available for this purpose. Thus, the aim of the present study was to establish a fast and reliable method for the differentiation between emetic/non-emetic strains by MALDI-TOF MS. Selected strains/isolates of the B. cereus group as well as other Bacillus spp. (total nâ¯=â¯121) were cultured on sheep blood agar for 48â¯h before analysis. Subsequently, the cultures were directly analyzed by MALDI-TOF MS without prior extraction steps. The samples were measured in the mass range of m/z 800-1800â¯Da. Using ClinProTools 3.0 statistical software and Flex analysis software (Bruker Daltonics GmbH, Bremen, Germany), a differentiation between emetic/non-emetic isolates was possible with a rate of correct identification of 99.1% by means of the evaluation of two specific biomarkers (m/z 1171 and 1187â¯Da).
Asunto(s)
Bacillus cereus/metabolismo , Depsipéptidos/biosíntesis , Microbiología de Alimentos , Bacillus cereus/genética , Bioensayo , Biomarcadores , Biomasa , Contaminación de Alimentos/análisis , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: Fruits and vegetables have increasingly been related to foodborne outbreaks. Besides surface contamination, a possible internalization of microorganisms into edible parts of plants during growth has already been observed. To examine an actual risk for the consumer, microbial contamination of the rind and pulp of 147 muskmelons from international trade was assessed using cultural and biochemical methods, polymerase chain reaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: One hundred percent of the rind samples [3.69-8.92 log colony forming units (CFU) g-1 ] and 89.8% of the pulp samples (maximum load 3.66 log CFU g-1 ) were microbiologically contaminated. Among the 432 pulp isolates, opportunistic and potentially pathogenic bacteria were identified, mainly Staphylococcus spp. (48.9%), Clostridium spp. (42.9%) and Enterobacteriaceae (27.9%). Salmonella spp., Escherichia coli and isolates of the Bacillus cereus group were found on the rind (1.4%, 0.7% and 42.9%, respectively) and in the pulp (0.7%, 1.4% and 4.7%). Clostridium perfringens was isolated from the rind of seven melons. CONCLUSION: The present study revealed a regularly occurring internal contamination of melons. Possible health risks for consumers because of an occurrence of microorganisms in melon pulp should be considered in future food safety assessments. © 2018 Society of Chemical Industry.
Asunto(s)
Bacterias/aislamiento & purificación , Cucumis melo/microbiología , Contaminación de Alimentos/análisis , Microbiología de Alimentos/economía , Frutas/economía , Bacterias/clasificación , Bacterias/genética , Bacterias/crecimiento & desarrollo , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Inocuidad de los Alimentos , Frutas/microbiologíaRESUMEN
In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.
Asunto(s)
Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/veterinaria , Sus scrofa/virología , Enfermedades de los Porcinos/diagnóstico , Animales , Animales Salvajes/virología , Ensayo de Inmunoadsorción Enzimática , Heces/virología , Hepatitis E/diagnóstico , Filogenia , Polonia , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Análisis de Secuencia de ARN , PorcinosRESUMEN
Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.
Asunto(s)
Proteínas Fúngicas/aislamiento & purificación , Extracción Líquido-Líquido/métodos , Micelio/clasificación , Stachybotrys/clasificación , Acetonitrilos/química , Formiatos/química , Micelio/química , Micelio/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Stachybotrys/química , Stachybotrys/crecimiento & desarrollo , Tricotecenos/biosíntesisRESUMEN
1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020-2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC-ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number.
Asunto(s)
Contaminación de Alimentos , Leche , Alcaloides de Pirrolicidina , Tropanos , Animales , Leche/química , Alcaloides de Pirrolicidina/análisis , Alemania , Tropanos/análisis , Bovinos , Contaminación de Alimentos/análisis , Granjas , Espectrometría de Masas en Tándem , Industria LecheraRESUMEN
Ragworts like tansy ragwort (J. vulgaris Gaertn., syn. Senecio jacobaea L.) contain hepatotoxic and cancerogenic pyrrolizidine alkaloids (PA) and their corresponding pyrrolizidine alkaloid N-oxides (PANO). Due to increasing spread of ragworts (Jacobaea spp.) PA/PANO may pose a health risk to animals and humans consuming contaminated feed and food. Therefore, the aim of the present study was to investigate the transfer of individual PA/PANO originating from a well-defined PA/PANO extract into the milk of dairy cows. For this objective, 16 German Holstein cows were assigned to four treatment groups (n = 4) in a 28-day dose-response study. Administration into the reticulorumen was performed daily by gavage after the morning milking. Three groups received different amounts of the J. vulgaris extract resulting in a PA/PANO exposure of 0.47, 0.95, or 1.91 mg PA/PANO/kg body weight/day, respectively. Furthermore, a control group received molasses to account for the sugar content of the used PA/PANO extract. While the composition of the PA/PANO extract was more diverse, the PA/PANO pattern in milk was dominated by the PA in their free base form. It was shown that mainly PA considered stable in the rumen environment were transferred into the milk. The main compounds in milk were jacoline (74.3 ± 2.4% of the PA/PANO sum), jaconine (11.2 ± 1.3%), and jacobine (7.2 ± 0.6%) with concentrations up to 29.7, 4.65 µg/l, or in the highest exposed group, 3.44 µg/l. There was no dose-dependent effect on the total PA/PANO transfer rate into the milk. The average transfer rate was 0.064 ± 0.005% of the administered content.
RESUMEN
SCOPE: 1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity. METHODS AND RESULTS: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na+ /taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA-mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time-dependent manner and attenuated PA-mediated cytotoxic effects in HepaRG cells. CONCLUSION: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Alcaloides de Pirrolicidina , Cationes/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos , Humanos , Péptidos/metabolismo , Alcaloides de Pirrolicidina/toxicidad , Ácido Taurocólico/metabolismoRESUMEN
Within the European Union (EU), edible insects need to be approved as "Novel Food" according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 µg/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.
Asunto(s)
Alimentación Animal/análisis , Larva/química , Larva/fisiología , Toxina T-2/análogos & derivados , Toxina T-2/análisis , Tenebrio/química , Animales , Fusarium/química , Fusarium/crecimiento & desarrollo , Fusarium/fisiología , Larva/crecimiento & desarrollo , Tenebrio/metabolismoRESUMEN
Culturing methods are conventionally applied to investigate the contamination of food with several microorganisms after heat processing. However, with these methods, it is not possible to evaluate whether heat-treated meat products, such as cooked sausages, contained parts of spoiled meat. Therefore, two specific multiplex qPCRs were developed in this study in order to determine the microbiological quality of the raw materials used for these products. The PCR targets focused on four bacterial groups often found on meat (family Enterobacteriaceae, genus Pseudomonas, genus Staphylococcus and species Brochothrix thermosphacta). Specificity as well as sensitivity of the developed multiplex qPCRs, validated by using 68 microbial species, were 100%. The applicability of both multiplex qPCRs compared to culturing methods was performed using 96 meat samples (fresh and naturally spoiled) and 12 inhouse-made "Lyoner" sausages containing variable ratios of spoiled meat (0%, 5%, 12% and 25%; n = 3 for each group). Both methods showed similar results by evaluating the ∆log10 cfu/g, the relative accuracy and the t-test analysis (p > 0.05). Comparing qPCR results of the different sausage groups, a significant difference between sausages containing fresh meat and sausages containing spoiled meat (12% and 25%) was found only for Pseudomonas and B. thermosphacta in both raw and cooked sausages. The statistical difference between 5% vs. 12% and 25% spoiled meat in cooked sausages, was also found only for these two bacterial groups. The developed multiplex qPCRs were further applied to 30 commercially available "Bologna-type" sausages. The results showed a total of 14 sausages considered to be suspicious for Food Fraud. While the role of Staphylococcus spp. in meat spoilage remains unclear, Pseudomonas, Enterobacteriaceae and B. thermosphacta could together be used as an indicator for "spoiled meat" used in sausages. The developed qPCR systems in this study allow the detection of four relevant bacterial groups in the heated Bologna-type sausages and provide information about the hygienic quality of raw materials used. This method could thus be helpful for screening food suspected of Food Fraud.
Asunto(s)
Bacterias/genética , Microbiología de Alimentos/métodos , Productos de la Carne/microbiología , Carne/microbiología , Reacción en Cadena de la Polimerasa , Animales , Brochothrix/genética , Enterobacteriaceae/genética , Calor , Pseudomonas/genética , Staphylococcus/genéticaRESUMEN
Pyrrolizidine alkaloids (PA) and their Noxides (PANO) are a group of toxic secondary plant metabolites occurring predominantly as contaminants in (herbal) teas, honeys and food supplements, as well as in spices and culinary herbs. Depending on the botanical origin of the contaminating plant, the pattern of PA/PANO can strongly vary within a sample. The current study aimed to broaden the existing data on the occurrence of PA/PANO in spices and culinary herbs. For this, 305 authentic samples covering 15 different matrices mainly harvested in 2016 or 2017 and originating from 36 countries were investigated for the presence of 44 PA/PANO. Fifty-eight percent of the samples contained at least one PA/PANO. The average sum content over all samples was 323 µg/kg (median of 0.9 µg/kg, 95% percentile of 665 µg/kg). The highest amount of 24.6 mg/kg was detected in an oregano sample. Additionally, conspicuous analyte patterns were discovered in samples from similar cultivation regions, indicating related botanical sources of PA/PANO contaminations. Particularly, oregano and cumin from Turkey often contained high amounts of PA/PANO. The results were used to assess the acute and chronic health risks related to PA/PANO intake via spices and culinary herbs, indicating a potential health risk in particular for adults and children with high consumption or when considering worstcase contamination scenarios of a sum content of 5500 µg/kg.