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The issue of food safety has acquired increased importance, and fraud is a major concern for the food industry. Among different types of food adulteration, there is the sale of frozen-thawed smoked salmon product as fresh, which not only decreases the quality of products but also misleads consumers and may involve associated health risks. In response to this problem, we tested the performance of histology to identify smoked salmon as fresh or frozen-thawed as a valid analytical method, so food business operators and official controllers can reliably and correctly classify the storage state of the product. Three groups of samples were prepared: group A (n = 36), fresh samples; group B (n = 36), frozen at −18°C for 30 days; and group C (n = 36), stored at −3°C for 30 days after packaging. Two histopathologists examined all samples in blind evaluations and classified them as fresh or frozen-thawed. Sensitivity, specificity, and interrater agreement were calculated. Results show high performance with the test: 80.6% sensitivity (95% confidence intervals [CI]: 64 to 91.8%); 95.6% specificity (95% CI: 89.1 to 98.8%); and Cohen's kappa was 0.81 (95% CI: 0.64 to 0.98%). Histology is a reliable and highly accurate method to differentiate fresh from frozen-thawed smoked salmon and could be used by the industry and official controllers to verify the labeling of the commercial product. [ABSTRACT FROM AUTHOR]

European Commission Regulation (EU) 1276/2011 requires that fishery products intended for raw consumption be frozen at --20°C for not less than 24 h or at --35°C for at least 15 h in order to kill viable parasites other than trematodes. But because marinating processes are not always effective in destroying nematode larvae, raw marinated fish preparations should be frozen before consumption. This study evaluated the performance of a standardized histological method to distinguish between fresh and frozen-thawed raw marinated fish. Sixty anchovy (Engraulis encrasicolus) fillets were sampled: 30 were marinated at -t-4°C for 24 h, and 30 were frozen at --20°C for 24 h before being marinated for 24 h. All 60 samples were fixed in formalin, processed for paraffin embedding, cut, and stained with hematoxylin and eosin. The slide preparations were examined microscopically by three independent histopathologists and classified as frozen-thawed or negative according to standard operating procedure criteria in use at our laboratory. Performance evaluation of the method showed 100% sensitivity (95% confidence interval [Cl], 88.4 to 100%) and 100% specificity (95% Cl, 88.4 to 100%), and the interrater agreement (Cohen's kappa) was 1 (95% Cl, 0.85 to 1). Histology proved a valid and reliable tool to distinguish fresh from frozen-thawed marinated fish. It can be applied to deliver safe raw fishery products to consumers in order to minimize the risk of anisakidosis. [ABSTRACT FROM AUTHOR]

Administering growth promoters to meat-producing animals is strictly regulated within the European Community to preserve consumers' safety. Nevertheless, illicit misuse of steroids and other veterinary drugs to increase animal production has still been found by National Residues Control Plans (NRCPs), mainly based on expensive targeted multi-class/multi-residue analytical methods, used for confirming quantification analysis of these residues in tissues and biological fluids. The setup of complementary novel diagnostic methods, based on indirect biomarkers of exposure to anabolic substances is, therefore, needed to update current tests available in NRCPs at the screening level, where cheaper, sensitive and untargeted methods are required. Biological effects of illicit treatment transcriptomic analysis has reached a good grade of standardization, identified by microarray and RNAseq technologies of large panels of differentially expressed gene targets (DEG) related to abuse of different classes of anabolic molecules (e.g. sex steroids, thyreostats, β 2 -agonists, glucocorticoids, etc.). The aim of the work was to identify a minimum set of molecular markers able to detect illicit administration of two of the most recurrent classes of growth promoters (sex steroids and B 2 -agonists) on formalin-fixed paraffin-embedded (FFPE) tissues, being a potentially straightforward sampling strategy at the slaughterhouse level. A PCR array to allow quantitative profiling of 48 genetic targets was, therefore, developed on FFPE liver samples from veal calves. Conventional univariate and alternative approaches based on multivariate classification methods were considered for gene expression profiling of 92 samples collected from different animal trials, where effects of nandrolone, estradiol, their combination, and clenbuterol were studied. The developed Partial Least Squares – Discriminant Analysis (PLS-DA) models show the successful setup of a novel screening strategy based on multiple transcriptional biomarkers, able to expand tested sample sizes, and consequently strengthen current NRCPs. [Display omitted] • Novel indirect detection of growth promoters residues in food is needed. • Transcriptional biomarkers profiling has reached a good grade of standardization. • A qPCR array was developed to work on FFPE samples collected in Italian NRCP. • Developed PLS-DA models allowed to setup alternative screening strategies. [ABSTRACT FROM AUTHOR]

• An available assay to detect sex-steroids abuse in calves was modified to work on FFPE. • Method validation was carried out on FFPE archive blocks from experimental trials. • A RT-qPCR comparative study of FFPE and RNAlater field samples was performed. • The modified assay may overcome the limitations of field sampling at slaughterhouse. In the context of mRNA biomarker profiling, formalin fixed paraffin embedded (FFPE) samples represent an interesting source for retrospective analysis. However, the implementation in routine analysis of FFPE samples, following legislation demands for validated and accredited methods, often requires critical revision and optimization. In the frame of an official control program the validation study of a molecular test for detection of sex steroids administration in calves, based on quantification of progesterone-Receptor mRNA in bulbo-urethral gland samples, was performed: analyses were made on FFPE tissues sections routinary used for histological investigations and compared with RNAlater tissue preservation. To overcome the limitations of original assays several modifications were tested. Obtained results confirmed how Progesterone-Receptor assay represent a useful tool to study suspected cases of sex steroid illicit administration in veal calves, complementary to histological and/or immune histochemical investigation, overcoming the limitation of field studies, where optimal pre-analytical condition cannot always be guarantee. [ABSTRACT FROM AUTHOR]

Abstract Fighting food frauds is a ceaseless challenge because of the constant evolution of fraudulent practices and for the consequences both on consumers' and on globalized trade. In Italy fish is a vulnerable commodity for frauds thanks to the high national production, importation and consumption and it is important to monitor the entire food chain in order to detect and prevent fraudulent actions, such as species substitutions, which is considered the most common fraud in seafood. Aim of this study was to realise a targeted Monitoring Plan to estimate the prevalence of fish species substitutions in Italy. As a first step, Italian fish supply chain, from production to selling, was analysed, in order to identify products and chain points at risk, by reviewing literature and by involving, in two focus groups, food inspectors and representatives of the large scale food distribution system. Then a monitoring plan was designed by sampling three fish species considered at major risk for their economic value and/or large consumption, i.e. tuna, grouper and flat fishes, at different selling points (wholesale markets, retail markets, fish shops and supermarkets). From February to March 2017, 242 samples from fresh, frozen or transformed whole fishes or fillets were sampled in 13 Italian cities (5 in Northern Italy, 2 in Central Italy, 3 in Southern Italy and 3 in the main Islands). Samples were analysed by "FINS" (Forensically Informative Nucleotide Sequencing), using two markers: cytochrome oxidase subunit I gene (COI) as first option and then mitochondrial cytochrome B gene (cytb), if necessary to identify uncertain or unassigned samples. Species substitutions were uncovered in 8.7% of analysed samples, principally related to grouper (prevalence of 14.71%). Supermarkets resulted the selling points with a major number of frauds (prevalence of 12.79%). Substituted species were taxonomically related to those declared on the label and no species harmful for consumers were detected. Results obtained can give to National Authorities a detailed frame of trends in fish substitution frauds in Italy, providing also relevant information to put into effect control measures. Highlights • Targeted Italian Monitoring Plan to estimate the prevalence of fish substitution frauds in Italy. • Results of the first National Monitoring Plans on fish substitutions. • Trends in fish substitution frauds in Italy giving information to put into effect control measures. [ABSTRACT FROM AUTHOR]

Abstract: Asbestos has been banned in many countries, including Italy. However, sources of exposure may still exist, due to asbestos in-situ or past disposal of asbestos-containing waste. In an urban area with past high environmental exposure, like Casale Monferrato, the lung fiber burden in sentinel animals may be useful to identify such sources. A pilot study was conducted to assess the feasibility of its determination in wild rats, a suitable sentinel species never used before for environmental lung asbestos fiber burden studies. Within the framework of pest control campaigns, 11 adult animals from 3 sites in the urban area of Casale Monferrato and 3 control rats from a different, unexposed town were captured. Further, 3 positive and 3 negative control lung samples were obtained from laboratories involved in breeding programs and conducting experimental studies on rats. Tissue fiber concentration was measured by scanning electron microscopy with energy dispersive spectrometry. Asbestos (chrysotile and crocidolite) was identified in the lungs from rats from Casale Monferrato, but not in control rats and in negative control lung samples. Asbestos grunerite at high concentration was found in positive control lung samples. Measurement of the lung fiber burden in wild rats has proved feasible: it was possible not only to detect, but also to characterize asbestos fibers both qualitatively and quantitatively. The pilot study provides the rationale for using wild rats as sentinels of the soil contamination level in Casale Monferrato, to identify areas with the possible presence of previously unrecognized asbestos sources. [Copyright &y& Elsevier]

Highlights: [•] Rapid dexamethasone urinary excretion was not related to the nature of the treatment. [•] The elimination rate of dexamethasone in tissues was slower than in urine. [•] Dexamethasone was widely distributed to the tissues. [•] Prednisolone residues in tissues suggest the endogenous nature of this molecule. [•] Prednisone was found only in the urine sample with the highest levels of prednisolone. [Copyright &y& Elsevier]

Abstract: The study was designed to evaluate anti-tumour properties of Iraqi propolis collected from Mosul region (M) on HL-60 and HCT-116 cell lines and on HCT-116 in vivo. M induced an inhibitory effect against the proliferation of HL-60 and colony potential of HCT-116 cells. The apoptosis in HL-60 cells was associated with down-regulation of Bcl-2 and activation of Bax, while in HCT-116 cells, necrotic features were observed; size of cells was dramatically increased by swelling of cytoplasm and loss of membrane integrity, cell rupture and release of cellular contents. Analysis of BrdU/DNA cell cycle in both cell lines showed that M induced cell cycle perturbations in both BrdU positive and BrdU negative cells. The exposure of HL-60 to M caused γ-H2AX in a dose dependent manner and was associated with induction of apoptosis. The experiments in HCT-116 tumor-bearing mice showed that oral administration of propolis at doses that caused no detectable toxicity was associated with a decrease in mitotic cells and an increase in endoreduplications, increased p53 and decreased Ki-67 expression of cells in tumor sections. This study provides the rationale to investigate the potential beneficial effect of propolis in the diet of patients receiving anti-cancer therapies. [Copyright &y& Elsevier]

Abstract: Natural and synthetic corticosteroids are widely used in veterinary medicine for their anti-inflammatory properties, but are also illegally used in animal breeding as growth-promoting agents: this latter application in livestock production has been banned within the European Union due to health concerns for the consumer. In this work urine samples collected from bovines experimentally treated with dexamethasone (0.4mg of dexamethasone 21-disodium phosphate per capita/day for 20 consecutive days) and bovines bred under strictly controlled conditions were investigated for the presence of natural and synthetic corticosteroids, using a simple multi-residue liquid chromatography–tandem mass spectrometry method, developed and validated in accordance with the criteria of the Commission Decision 2002/657/EC. The aim of this work is to investigate the effect of a low dosage and long term dexamethasone treatment on the levels of endogenous corticosteroids in cattle and to evaluate the possible presence of prednisolone residues in bovines bred under strictly controlled conditions. Our findings confirm the high and rapid rate of dexamethasone urinary excretion. Dexamethasone treatment elicited an early reduction of hydrocortisone and cortisone, suggesting the disappearance of these two hormones as an indirect indicator of corticosteroid treatment in cattle. Prednisolone residues were found (concentration interval 0.4–1.4ngmL−1) in urine samples collected from control bovines especially at the slaughterhouse, together with high levels of hydrocortisone and cortisone. Further studies are necessary to find out the reason of unexplained excretion of this hormone in urine samples of untreated bovines. [Copyright &y& Elsevier]

• Metabolomics analysis underscored markers of clenbuterol administration. • Clenbuterol administration affect metabolic pathways in liver. • Quantification of liver metabolites indirectly reveal clenbuterol administration. • A diagnostic tool to detect abuse of clenbuterol was developed and validated. Surveillance of illegal use of growth promoters such as β 2 -agonists in food producing animals rely on the detection of drug residues by LC-MS/MS. Screening strategies focusing on indirect physiological responses following administration of active compounds are promising approaches to strengthen existing targeted methods and ensure food safety. A metabolomics analysis based on LC-HRMS was carried out on liver extracts from bulls experimentally treated with clenbuterol combined with dexamethasone (n = 8) to mimic a potential anabolic practice, and control animals (n = 8). Nicotinic acid and 5′-deoxy-5′-methylthioadenosine were identified as biomarkers of treatment. Ratio values of such markers to others of the same metabolic pathways (nicotinamide or methionine) were used to develop a classification model to assign animals as treated with clenbuterol or non-treated. The classification model was tested on an external validation set comprising 74 animals either treated with different anabolic compounds (β 2 -agonists, sexual steroids, corticosteroid), or non-treated, showing 100% sensitivity and specificity. [ABSTRACT FROM AUTHOR]

• This paper describe the first in vivo trial supporting bile as matrix for DXM detection at slaughterhouse. • DXM induces severe thymic atrophy in Chianina beef bull identifiable through histology and morphometry. • Results obtained by bile analyses are supported by histological study of thymus. Dexamethasone (DXM) is a synthetic adrenal corticosteroid with anti-inflammatory properties used for therapeutic purposes in a wide range of pathologies and of the most common corticosteroids used for anabolic purposes in beef cattle. It is proven that DXM induces histological changes, traceable as increasing fatty infiltration of the thymus associated with a concurrent decrease of the cortex-medulla ratio, so the histological examination of the thymus gland has been established as an indirect morphological biomarker. The aim of the present study is to compare thymus histology and DXM concentrations in biological fluids collected at slaughterhouse after 1 month of DXM treatment. Our findings demonstrate that a low dosage of DXM administered to 12 months-old-Chianina beef cattle induces severe thymic atrophy with concurrent reduction of the cortex/medulla ratio, demonstrable even when DXM residues are not found in serum and urine samples. It is worth to note that, at the slaughterhouse, DXM residues are detectable in bile samples, indicating the ability of this biological fluid to bio-concentrate the administered drug if compared to serum and urine. Therefore, bile could be candidates as new liquid matrix for the screening programs planned to contrast the illegal use of anabolic substances. [ABSTRACT FROM AUTHOR]