Fabiola Pizzo

Individual and combined effects of deoxynivalenol and α-zearalenol on cell proliferation and steroidogenesis of granulosa cells in cattle

This study was conducted to evaluate the impact of deoxynivalenol (DON) and zearalenone (ZEA) metabolite, α-zearalenol (α-Zol), on cell proliferation and steroidogenesis of bovine large (LG) follicle granulosa cells (GC). LGGC were obtained from bovine ovarian follicles (8-22 mm) and were cultured for 2 days in medium containing 10% fetal bovine serum followed by 1 or 2 days in serum-free medium without (control) or with treatments. Three different experiments were performed using different dosages of DON and α-Zol and in different combinations and a fourth experiment evaluated estradiol effects on granulosa cell proliferation. DON inhibited progesterone (P4) and estradiol (E2) production at high dose. α-Zol alone and in combination with DON increased cell growth. Estradiol inhibited cell growth indicating α-Zol is not acting as an estrogen agonist. This study demonstrates that α-Zol and DON can impact in vitro GC function, however further studies will be required to better understand the mechanism of action and reproductive effects of Fusarium mycotoxins.

Epidemiological study (2006–2012) on the poisoning of small animals by human and veterinary drugs

A retrospective study was conducted on the exposure of dogs and cats to drugs, reported to the Poison Control Centre of Milan (Centro Antiveleni di Milano (CAV)) between January 2006 and December 2012. Calls related to drugs for human use and veterinary drugs accounted for 23.7 per cent of total inquiries (1415) received by CAV and mostly involved dogs (70 per cent of enquiries). Exposure to drugs for human use accounted for 79 per cent of cases involving dogs, whereas veterinary drugs were the main culprit (77 per cent) in the case of cats. The most common class of drugs for human use proved to be CNS drugs (26.8 per cent), followed by NSAIDs (19.6 per cent) and cardiovascular and endocrine drugs (12.9 per cent each). The majority of calls (95.2 per cent) related to veterinary drugs involved dogs and cats exposed to parasiticides. The outcome was reported in only 58.2 per cent of cases, and fatal poisoning accounted for 8.7 per cent of these cases. Epidemiological data from this Italian survey provide useful information on animal exposure to drugs. The knowledge of agents involved in poisoning episodes can help veterinarians make the correct diagnosis and institute preventive measures to possibly reduce animal exposure to drugs.

Poisoning of dogs and cats by drugs intended for human use.

One of the main causes of poisoning of small animals is exposure to drugs intended for human use. Poisoning may result from misuse by pet owners, off-label use of medicines or, more frequently, accidental ingestion of drugs that are improperly stored. This review focuses on classes of drugs intended for human use that are most commonly involved in the poisoning of small animals and provides an overview of poisoning episodes reported in the literature. To perform this review a comprehensive search of public databases (PubMed, Web of Science, Scopus, Google Scholar) using key search terms was conducted. Additionally, relevant textbooks and reference lists of articles pertaining to the topic were reviewed to locate additional related articles. Most published information on small animal poisoning by drugs intended for human use was from animal and human poison control centres or from single case reports. The dog was the species most frequently poisoned. The major drugs involved included analgesics (nonsteroidal anti-inflammatory drugs), antihistamines (H1-antihistamines), cardiovascular drugs (calcium channel blockers), central nervous system drugs (selective serotonin reuptake inhibitors, baclofen, benzodiazepines and zolpidem), gastrointestinal drugs (loperamide), nutritional supplements (vitamin D and iron salts) and respiratory drugs (β2-adrenergic receptor agonists).

Transport of Aflatoxin M1 in Human Intestinal Caco-2/TC7 Cells

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1). After it is formed, it is secreted in the milk of mammals. Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity, and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions (TJ) of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively. In this study, the Caco-2/TC7 cells were treated with different AFM1 concentrations (10–10,000 ng/kg) for short (40 min) and long periods of time (48 h). The AFM1 influx/efflux transport and effects on TJ were evaluated by measuring trans-epithelial electrical resistance and observing TJ protein (Zonula occludens-1 and occludin) localization. The results showed that: (i) when introduced to the apical and basolateral compartments, AFM1 was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (Papp value of 105.10 ± 7.98 cm/s × 10−6). (ii) The integrity of TJ was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either. The present results contribute to the evaluation of human risk exposure to AFM1, although the AFM1 transport mechanism need to be clarified.

A New Structure-Activity Relationship (SAR) Model for Predicting Drug-Induced Liver Injury, Based on Statistical and Expert-Based Structural Alerts

The prompt identification of chemical molecules with potential effects on liver may help in drug discovery and in raising the levels of protection for human health. Besides in vitro approaches, computational methods in toxicology are drawing attention. We built a structure-activity relationship (SAR) model for evaluating hepatotoxicity. After compiling a data set of 950 compounds using data from the literature, we randomly split it into training (80%) and test sets (20%). We also compiled an external validation set (101 compounds) for evaluating the performance of the model. To extract structural alerts (SAs) related to hepatotoxicity and non-hepatotoxicity we used SARpy, a statistical application that automatically identifies and extracts chemical fragments related to a specific activity. We also applied the chemical grouping approach for manually identifying other SAs. We calculated accuracy, specificity, sensitivity and Matthews correlation coefficient (MCC) on the training, test and external validation sets. Considering the complexity of the endpoint, the model performed well. In the training, test and external validation sets the accuracy was respectively 81, 63, and 68%, specificity 89, 33, and 33%, sensitivity 93, 88, and 80% and MCC 0.63, 0.27, and 0.13. Since it is preferable to overestimate hepatotoxicity rather than not to recognize unsafe compounds, the models architecture followed a conservative approach. As it was built using human data, it might be applied without any need for extrapolation from other species. This model will be freely available in the VEGA platform.

Integrated in silico strategy for PBT assessment and prioritization under REACH

Chemicals may persist in the environment, bioaccumulate and be toxic for humans and wildlife, posing great concern. These three properties, persistence (P), bioaccumulation (B), and toxicity (T) are the key targets of the PBT-hazard assessment. The European regulation for the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) requires assessment of PBT-properties for all chemicals that are produced or imported in Europe in amounts exceeding 10 tonnes per year, checking whether the criteria set out in REACH Annex XIII are met, so the substance should therefore be considered to have properties of very high concern. Considering how many substances can fall under the REACH regulation, there is a pressing need for new strategies to identify and screen large numbers fast and inexpensively. An efficient non-testing screening approach to identify PBT candidates is necessary, as a valuable alternative to money- and time-consuming laboratory tests and a good start for prioritization since few tools exist (e.g. the PBT profiler developed by US EPA). The aim of this work was to offer a conceptual scheme for identifying and prioritizing chemicals for further assessment and if appropriate further testing, based on their PBT-potential, using a non-testing screening approach. We integrated in silico models (using existing and developing new ones) in a final algorithm for screening and ranking PBT-potential, which uses experimental and predicted values as well as associated uncertainties. The Multi-Criteria Decision-Making (MCDM) theory was used to integrate the different values. Then we compiled a new set of data containing known PBT and non-PBT substances, in order to check how well our approach clearly differentiated compounds labeled as PBT from those labeled as non-PBT. This indicated that the integrated model distinguished between PBT from non-PBT compounds.

Direct effects of the algal toxin, domoic acid, on ovarian function: Bovine granulosa and theca cells as an in vitro model

Domoic acid (DA) is a potent neurotoxin produced by alga Pseudo-nitzschia spp. and has been associated with reproductive disorders in mammals. The aim of this study was to investigate if DA can affect the reproductive system via direct action on ovarian function. Bovine granulosa and theca cells were used as in vitro models for evaluating DA effects on ovarian cell proliferation and steroid production. In small-follicle granulosa cells (SMGC), cell proliferation and estradiol (E2) production was not affected (P>0.05) while progesterone (P4) production was inhibited (P<0.05) by DA at all doses tested. In large-follicle granulosa cells (LGGC), DA had no effect (P>0.05) on cell proliferation or P4 production while E2 production was stimulated by 1 and 5 µg/ml DA (P<0.05). DA (1 µg/ml) attenuated (P<0.05) insulin-like growth factor 1 (IGF-1)-induced P4 production by large-follicle theca cells (LGTC), but did not affect androstenedione (A4) production or proliferation of LGTC. In glutamate-free medium, DA inhibited (P<0.05) SMGC E2 production and this inhibition was similar to inhibition of E2 by trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid monohydrate (ACPD; a selective metabotropic glutamate receptor subtype agonist) while kainic acid (KA; an ionotropic glutamate receptor subtype agonist) had no effect (P>0.10) on E2 production. Collectively, these results show for the first time that DA has direct effects on ovarian GC and TC steroidogenesis. Because DA inhibited E2 and P4 production, DA has the potential to be an endocrine disruptor.

In Silico Models for Repeated-Dose Toxicity (RDT): Prediction of the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) for Drugs.

The preclinical stage in drug development requires the determination of repeated-dose toxicity (RDT) in animal models. The main outcome of RDT studies is the determination of the no observed adverse effect level (NOAEL) and the lowest observed adverse effect level (LOAEL). NOAEL is important since it serves to calculate the maximum recommended starting dose (MRSD) which is the safe starting dose for clinical studies in human beings. Since in vivo RDT studies are expensive and time-consuming, in silico approaches could offer a valuable alternative. However, NOAEL and LOAEL modeling suffer some limitations since they do not refer to a single end point but to several different effects and the doses used in experimental studies strongly influence the final results. Few attempts to model NOAEL and LOAEL have been reported. The available database and models for the prediction of NOAEL and LOAEL are reviewed here.

Evaluation of di‐magnesium malate, used as a novel food ingredient and as a source of magnesium in foods for the general population, food supplements, total diet replacement for weight control and food for special medical purposes

Abstract The present scientific opinion deals with the evaluation of the safety of di‐calcium malate (DCM) proposed as a novel food ingredient and as a source of calcium for use in foods for the general population, food supplements, total diet replacement for weight control and food for special medical purposes (FSMP), and with the bioavailability of calcium from this source. The structural formula of the proposed complex is based on expert judgement and not supported by any analytical data. On the basis of the available data, the Panel concluded that there was insufficient scientific evidence of a difference between the proposed novel food ingredient named as di‐calcium malate (DCM) and calcium malate already authorised as a source of calcium included in Annex II to Directive 2002/46/EC. Accordingly, the Panel was unable to assess the safety of DCM as a novel food ingredient. On the basis of the results provided, the Panel considered that DCM does not completely dissociate into calcium and malic acid. The Panel concluded that when DCM dissociates, calcium would be available following ingestion of DCM and the bioavailability would appear similar to values reported for other sources of calcium already permitted. Furthermore, the Panel concluded that on the basis of the information available it was not possible to calculate the exposure to DCM as a source of calcium to foods for the general population, food supplements, total diet replacement for weight control and FSMP.

Scientific opinion on the safety of monacolins in red yeast rice

Abstract The Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on the safety of monacolins in red yeast rice (RYR) and to provide advice on a dietary intake of monacolins that does not give rise to concerns about harmful effects to health. The Panel reviewed the scientific evidences available as well as the information provided by interested parties in response of a public ‘Call for data’ launched by EFSA. The Panel considered that monacolin K in lactone form is identical to lovastatin, the active ingredient of several medicinal products authorised for the treatment of hypercholesterolaemia in the EU. On the basis of the information available, the Panel concluded that intake of monacolins from RYR via food supplements, could lead to estimated exposure to monacolin K within the range of the therapeutic doses of lovastatin. The Panel considered that the available information on the adverse effects reported in humans were judged to be sufficient to conclude that monacolins from RYR when used as food supplements were of significant safety concern at the use level of 10 mg/day. The Panel further considered that individual cases of severe adverse reactions have been reported for monacolins from RYR at intake levels as low as 3 mg/day. The Panel concluded that exposure to monacolin K from RYR could lead to severe adverse effects on musculoskeletal system, including rhabdomyolysis, and on the liver. In the reported cases, the product contained other ingredients in addition to RYR. However, these reported effects in particular musculoskeletal effects, have both occurred after ingestion of monacolin K and lovastatin independently. On the basis of the information available and several uncertainties highlighted in this opinion, the Panel was unable to identify a dietary intake of monacolins from RYR that does not give rise to concerns about harmful effects to health, for the general population, and as appropriate, for vulnerable subgroups of the population.