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Mycotoxins are chemical contaminants that are invisible, tasteless, chemically stable and survive food processing. Contamination along the agri-food chain is difficult to control since their production and spreading are due to numerous factors including temperature, relative humidity, insect infestation, and susceptibility of the host plant. This is a pilot study which aims at assessing the contamination level of deoxynivalenol (DON), and its plant metabolites (3AcDON, 15 AcDON, DON 3G), nivalenol, T-2 and HT-2 toxins, and ochratoxin A in thirty-seven traditional varieties of Ethiopian durum wheat, and aflatoxins B1, B2, G1, and G2 in thirty-one varieties of Ugandan groundnuts grown in non-intensive rainfed production systems. Results indicate absence of mycotoxin contamination in all durum wheat samples and negligible levels of contamination (below the maximum levels tolerated by international standards) in groundnut samples. Further studies are required to assess if non-intensive production systems and varieties have a role in preventing and/or reducing mycotoxin contamination of the crops.

Genus Claviceps is a plant pathogen able to produce a group of toxins, ergot alkaloids (EAs), whose effects have been known since the Middle Ages (ergotism). Claviceps purpurea is the most important representative specie, known to infect more than 400 monocotyledonous plants including economically important cereal grains (e.g., rye, wheat, triticale). EAs are not regulated as such. Maximum limits are in the pipeline of the EU Commission while at present ergot sclerotia content is set by the Regulation (EC) No. 1881/2006 in unprocessed cereals (0.05% as a maximum). This study aimed to investigate the presence of the six principal EAs (ergometrine, ergosine, ergocornine, &alpha, ergocryptine, ergotamine and ergocristine) and their relative epimers (-inine forms) in rye- and wheat-based products. Of the samples, 85% resulted positive for at least one of the EAs. Wheat bread was the product with the highest number of positivity (56%), followed by wheat flour (26%). Rye and wheat bread samples showed the highest values when the sum of the EAs was considered, and durum wheat bread was the more contaminated sample (1142.6 &mu, g/kg). These results suggest that ongoing monitoring of EAs in food products is critical until maximum limits are set.

Ochratoxin A is one of the most diffused mycotoxin present in a large spectrum of food commodities, mainly produced by Aspergillus ochraceus, Aspergillus carbonarius, Aspergillus niger and Penicillium verrucosum. EU has set maximum limits for a number of matrices such as cereals, wine, spices and liquorice, whilst other commodities such as beer and meat products that are susceptible of OTA contamination and are largely consumed are not included. In 2013, within the framework of the Regulation (EC) 882/2004 on official controls, the European Commission issued the mandate M/520 regarding the standardisation for methods of analysis for mycotoxins in food to the European Committee for Standardisation. Of the 11 priorities of the mandate, the one on “HPLC determination of OTA in meat, meat products and edible offal” was assigned to the Italian National Reference Laboratory for feed and food. The method was single-laboratory validated, and all the performance characteristics of the method were compliant with the corresponding reference values indicated in Regulation (EC) n. 401/2006. The method was applied to characterise a set of 5 pork-based materials (ham, kidney, liver and canned chopped pork) to be used for an inter-laboratory method validation study. Three ham materials (levels of contamination of 0.77, 2.22 and 12.3 μg/kg, respectively), one liver material (contamination level of 2.80 μg/kg) and one chopped pork meat (contamination level of 0.66 μg/kg) were tested for homogeneity and stability.

Developmental health risks of chronical exposure to low doses of foodborne persistent organic pollutants (POP) are recognized but still largely uncharacterized. Juvenile female BALB/c mice exposed to either HBCD, CB-153 or TCDD at doses relevant to human dietary exposures (49.5 μg, 1.35 μg and 0.90 ng kg−1 bw−1 day−1, respectively) for 28 days displayed histopathological changes in liver (HBCD, CB-153, TCDD), thymus (HBCD, CB-153) and uterus (HBCD), reduced serum oestradiol 17β (E2) levels (HBCD), increased serum testosterone (T) levels (CB-153) and an increased T/E2 ratio (HBCD). Proteomics analysis of brain provided molecular support for the HBCD-induced reduction in E2. Neural gene expression analysis, confirmed effects on 18 out of 30 genes previously found to be affected after exposure to higher doses to the same pollutants. Our findings indicate that exposure to POP at low doses is associated with subtle, but toxicological relevant effects on post-natal development in female mice.

The analytical scenario for determining contaminants in the food and feed sector is constantly prompted by the progress and improvement of knowledge and expertise of researchers and by the technical innovation of the instrumentation available. Mycotoxins are agricultural contaminants of fungal origin occurring at all latitudes worldwide and being characterized by acute and chronic effects on human health and animal wellness, depending on the species sensitivity. The major mycotoxins of food concern are aflatoxin B1 and ochratoxin A, the first for its toxicity, and the second for its recurrent occurrence. However, the European legislation sets maximum limits for mycotoxins, such as aflatoxin B1, ochratoxin A, deoxynivalenol, fumonisins, and zearalenone, and indicative limits for T-2 and HT-2 toxins. Due to the actual probability that co-occurring mycotoxins are present in a food or feed product, nowadays, the availability of reliable, sensitive, and versatile multi-mycotoxin methods is assuming a relevant importance. Due to the wide range of matrices susceptible to mycotoxin contamination and the possible co-occurrence, a multi-mycotoxin and multi-matrix method was validated in liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose to overcome specific matrix effects and analyze complex cereal-based samples within the Italian Total Diet Study project.

Ethylenethiourea (ETU) is the common metabolite of the widely used ethylenebisdithiocarbamate fungicides. It is identified as Endocrine Disruptor given its ability to interfere with thyroid hormone biosynthesis by inhibiting thyroid peroxidase activity. As far as we know, no studies have been performed to assess potential effects of ETU exposure at low dose levels, i.e. below the established LOAEL and NOAEL, during critical phases of development. Therefore, the aim of the present study was to verify the short- and long-term effects on thyroid function, reproduction and development of oral exposure to ETU levels comparable to and lower than LOAEL/NOAEL in rats. Sixty dams were treated daily by gavage during pregnancy and lactation with 0, 0.1, 0.3, 1.0 mg/kg bw per day of ETU. F1 generation was similarly treated from weaning to sexual maturity. Thyroid biomarkers were analyzed in dams and in offspring. Reproductive biomarkers were analyzed in F1 rats. For the first time this study has demonstrated reproductive toxicity and hypothyroidism at a lower than LOAEL dose exposure in pregnant dams and F1 generation. Our data suggest that even low doses of ETU can interfere with thyroid homeostasis and reproductive hormone profile if exposure starts in critical stages of development.

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2′,4,4′-Tetrabromodiphenyl ether BDE-47; 2,2′,4,4′,5,5′-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 μg/kg bw/day; PCB-153 195 μg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects.

Environmental factors and genetic susceptibility are implicated in the increased risk of autism spectrum disorder (ASD). Mycotoxins are agricultural contaminants of fungal origin that represent real risk factors for human health and especially for children. Thus, the main hypothesis of this work is that the deterioration of the clinical manifestation of autism in children may result from the exposure to mycotoxins through the consumption of contaminated food. Within a cross-sectional study, a group of autistic children (n = 172) and a group of controls (n = 61) (siblings and non-parental) were recruited in North and South Italy. All children had blood and urine samples taken, for testing some mycotoxins by a LC-MS/MS validated method. Blood samples were also tested for assessing specific IgG against food and fungal antigens and cytokines. The analyses outputs highlighted statistically significant differences comparing mycotoxins levels between (i) children groups both in urine (deoxynivalenol and de-epoxydeoxynivalenol, p = 0.0141 and p = 0.0259, respectively) and serum (aflatoxin M1, ochratoxin A and fumonisin B1, p = 0.0072, p = 0.0141 and p = 0.0061, respectively); (ii) a group of selected fungal IgGs, and IgGs against wheat and gluten and (iii) cytokines. These results suggest the need for a deeper examination of the role that mycotoxins may have on the etiology of ASD.

Organophosphorus insecticides, as Chlorpyrifos (CPF), are widely used in agriculture and against household pests; these compounds receive an increasing consideration as potential endocrine disrupters. The aim of the present study was to examine the potential short- and long-term effects of CPF on thyroid and adrenal glands in CD1 mice following exposure at dose levels not inducing brain acetyl cholinesterase (AchE) inhibition, during gestational and/or postnatal vulnerable phases. Pregnant dams were treated with 0, 3, 6 mg/kg bw/day of CPF on gestational days 15-18. After delivery, pups were treated subcutaneously on postnatal days (PND) 11-14 with: 0, 1, 3 mg/kg bw/day of CPF. Serum thyroxin (T4), thyroid and adrenals histology and histomorphometry were evaluated in dams and in F1 mice. In dams at 6 mg/kg, decreased T4 levels and increased cell height in thyroid were observed, and adrenal histology showed a slightly increased vacuolization in the X-zone. In the F1, short-term morphological modifications (reduced follicular size at PND 2) and long-term morphological (increased necrotic follicular cells) and biochemical alterations (reduced serum T4 levels) were found at PND 150 with an apparent higher vulnerability of males. For the first time these results indicate that CPF exposure at dose levels not inducing brain AchE inhibition causes thyroid alterations in dams and in F1 CD1 mice. Thyroid may be a sensitive target to CPF developmental exposure possibly leading to long-term effects on thyroid function. Because thyroid plays a pivotal role in mammalian development, these findings can be relevant to humans.

Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods, in bleaching steps and flour treatment. Experimental data showed SEM acting as osteolathyrogen agent, but few toxicological data are available in susceptible life-stages. This study aimed to evaluate effects of SEM oral administration for 28 days at 0, 40, 75, 140 mg/kg bw day during the juvenile period in Sprague-Dawley rats. Histopatological examinations of: epiphyseal cartilage - potential target of SEM lathyrogen action - testes, ovary, uterus, thyroid, thymus, spleen, adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were performed. Mortality at high and mid dose levels and significantly decreased body weight gain were observed in males even at the lowest dose. Lack of mineralization in cartilage at all dose levels was present. Marked alterations of spontaneous motor and exploratory behaviours were evident even at 40 mg/kg. Histological alterations were observed in all tissues; thyroid and ovary effects were present also at 40 mg/kg. The present study indicate that the NOAEL in juvenile rats is lower than 40 mg/kg for SEM oral administration. SEM administration during juvenile period exerted pleiotropic effects and further studies are suggested to elucidate mechanisms.

The study explored possible reproductive and endocrine effects of short-term (5 days) oral exposure to anatase TiO2 nanoparticles (0, 1, 2 mg/kg body weight per day) in rat. Nanoparticles were characterised by scanning electron microscopy (SEM) and transmission electron microscopy, and their presence in spleen, a target organ for bioaccumulation, was investigated by single-particle inductively coupled plasma mass spectrometry and SEM/energy-dispersive X-ray. Analyses included serum hormone levels (testosterone, 17-β-estradiol and triiodothyronine) and histopathology of thyroid, adrenals, ovary, uterus, testis and spleen. Increased total Ti tissue levels were found in spleen and ovaries. Sex-related histological alterations were observed at both dose levels in thyroid, adrenal medulla, adrenal cortex (females) and ovarian granulosa, without general toxicity. Altered thyroid function was indicated by reduced T3 (males). Testosterone levels increased in high-dose males and decreased in females. In the spleen of treated animals TiO2 aggregates and increased white pulp (high-dose females) were detected, even though Ti tissue levels remained low reflecting the low doses and the short exposure time. Our findings prompt to comprehensively assess endocrine and reproductive effects in the safety evaluation of nanomaterials.

The plasticizer di-(2-ethylhexyl)phthalate (DEHP) affects reproductive development, glycogen and lipid metabolism. Whereas liver is a main DEHP target in adult rodents, the potential impact on metabolic programming is unknown. Effects of in utero DEHP exposure on liver development were investigated upon treatment of pregnant CD-1 mice on gestational days (GD)11-19. F1 mice were examined at post-natal days 21 (weaning) and 35 (start of puberty): parameters included liver histopathological, immunocytochemical and alpha-fetoprotein (AFP) gene expression analyses. In utero DEHP exposure altered post-natal liver development in weanling mice causing significant, dose-related (i) increased hepatosteatosis, (ii) decreased glycogen storage, (iii) increased beta-catenin intracytoplasmic localization (females only). At puberty, significantly decreased glycogen storage was still present in males. A treatment-induced phenotype was identified with lack of glycogen accumulation and intracytoplasmic localization of beta-catenin which was associated with increased AFP gene expression. Our findings suggested that DEHP alters post-natal liver development delaying the programming of glycogen metabolism.

The synthetic estrogen diethylstilbestrol (DES) is a model to study the effects on female reproductive tract of endocrine disrupting chemicals interacting with estrogen receptors. Pregnant CD-1 mice were given daily by gavage 10microg/kg bw of DES (the lower range of therapeutic exposure) during gestational days 9-16, critical period for reproductive tract development. Parameters of sexual development were recorded after weaning and at sexual maturation. No signs of general toxicity were observed in dams. In DES-treated group, reduced litter weight during lactation and earlier vaginal patency was observed. Uterus weight was increased in F1 treated females at weaning. Histological analysis showed reduced endometrium thickness and increased polyovular follicles, irregular and oocytes with condensed chromatin in the ovary at sexual maturity. Prenatal DES oral administration induces subtle but significant effects on puberty onset, uterine and ovary morphology.

Semicarbazide (SEM) is a food contaminant, by-product of azodicarbonamide sealing for gaskets of jar lids for baby foods. Toxicological data on SEM are insufficient; studies in rodents showed osteolathyrogenic effects in foetuses and potential carcinogenicity, but not genotoxicity. To characterize hazards for children, juvenile Sprague-Dawley rats of both sexes are exposed po to SEM from PND 23 to PND 60 at dose levels of 0 (vehicle only – distilled water), 40, 75 and 140 mg/kg bw day. During the treatment, body weight gain and food consumption are recorded; vaginal opening and preputial separation are observed to evaluate the development of reproductive system. At sexual maturity rats are sacrificed and tissues/organs are collected for histological analysis, with attention to endocrine, immune and skeletal systems: thyroid, adrenals, testes, spleen, uteri and ovaries, thymus, coxal-femoral joint. Body weight gain is reduced in males at 75–140 mg/kg. Food consumption is reduced in both sexes at 140 mg/kg and increased in females at 75 mg/kg. Vaginal opening is delayed in all treated groups; preputial separation is anticipated at 40 and 75 mg/kg and delayed in the high dose group. Histology of thyroid showed in all treated males increased desquamation in follicular lumen; in females this is present only at 140 mg/kg. Adrenal alterations are present in females only: cortex vacuolization (all dose groups) and blurred zonation architecture (high dose only), in medulla increased cell degeneration (high dose). In the spleen, increased haematopoiesis is present in males (high dose), with a dose-related trend at mid-dose. Such preliminary observations show alterations in endocrine and immune tissues. Altered timing in sexual maturation, adrenal cortex and thyroid effects are present also at 40 mg/kg, without evident general toxicity, supporting the need for toxicological studies on food and environmental contaminants targeted to juvenile lifestages.