Zootoxins and Domestic Animals: A European View.

Zootoxins are produced by venomous and poisonous species and are an important cause of poisoning in companion animals and livestock in Europe. Little information about the incidence of zootoxin poisoning is available in Europe, with only a few case reports and review papers being published. This review presents the most important zootoxins produced by European venomous and poisonous animal species responsible for poisoning episodes in companion animals and livestock. The main zootoxin-producing animal species, components of the toxins/venoms and their clinical effects are presented. The most common zootoxicoses involve terrestrial zootoxins excreted by the common toad, the fire salamander, the pine processionary caterpillar, and vipers. The lack of a centralized reporting/poison control system in Europe makes the evaluation of the epidemiology of zootoxin-induced poisonings extremely difficult. Even if there are many anecdotal reports in the veterinary community about the exposure of domestic animals to terrestrial and marine zootoxins, the number of published papers regarding these toxicoses is low. Climate change and its consequences regarding species distribution and human-mediated transportation are responsible for the emerging nature of some intoxications in which zootoxins are involved. Although new venomous or poisonous animal species have emerged in regions where they were previously unreported, zootoxins produced by native species remain the main concern in Europe. The diversity of poisonous and venomous animal species and the emerging nature of certain poisonings warrant the continuous update to such knowledge by veterinary professionals and animal owners. This review offers an overview about zootoxin-related poisonings in domestic animals in Europe and also provides important information from a health perspective.

Guar gum as a microbially degradable component for an oral colon delivery system based on a combination strategy: formulation and in vitro evaluation.

Oral colon delivery has widely been pursued exploiting naturally occurring polysaccharides degraded by the resident microbiota. However, their hydrophilicity may hinder the targeting performance. The aim of the present study was to manufacture and evaluate a double-coated delivery system leveraging intestinal microbiota, pH, and transit time for reliable colonic release. This system comprised a tablet core, a hydroxypropyl methylcellulose (HPMC) inner layer and an outer coating based on Eudragit® S and guar gum. The tablets were loaded with paracetamol, selected as a tracer drug because of the well-known analytical profile and lack of major effects on bacterial viability. The HPMC and Eudragit® S layers were applied by film-coating. Tested for in vitro release, the double-coated systems showed gastroresistance in 0.1 N HCl followed by lag phases of consistent duration in phosphate buffer pH 7.4, imparted by the HPMC layer and synergistically extended by the Eudragit® S/guar gum one. In simulated colonic fluid with fecal bacteria from an inflammatory bowel disease patient, release was faster than in the presence of ß-mannanase and in control culture medium. The bacteria-containing fluid was obtained by an experimental procedure making multiple tests possible from a single sampling and processing run. Thus, the study conducted proved the feasibility of the delivery system and ability of guar gum to trigger release in the presence of colon bacteria without impairing the barrier properties of the coating. Finally, it allowed an advantageous simulated colonic fluid preparation procedure to be set up, reducing the time, costs, and complexity of testing and enhancing replicability.

Emerging Plant Intoxications in Domestic Animals: A European Perspective.

Exposure to phytotoxins that are present in imported ornamental or native plants is an important cause of animal disease. Factors such as animal behaviors (especially indoor pets), climate change, and an increase in the global market for household and ornamental plants led to the appearance of new, previously unreported plant poisonings in Europe. This has resulted in an increase in the incidence of rarely reported intoxications. This review presents some of the emerging and well-established plant species that are responsible for poisoning episodes in companion animals and livestock in Europe. The main plant species are described, and the mechanism of action of the primary active agents and their clinical effects are presented. Data reflecting the real incidence of emerging poisoning cases from plant toxins are scarce to nonexistent in most European countries due to a lack of a centralized reporting/poison control system. The diversity of plant species and phytotoxins, as well as the emerging nature of certain plant poisonings, warrant a continuous update of knowledge by veterinarians and animal owners. The taxonomy and active agents present in these plants should be communicated to ensure awareness of the risks these toxins pose for domestic animals.

Methylglyoxal-induced neurotoxic effects in primary neuronal-like cells transdifferentiated from human mesenchymal stem cells: Impact of low concentrations.

In the last decades, advanced glycation end-products (AGEs) have aroused the interest of the scientific community due to the increasing evidence of their involvement in many pathophysiological processes including various neurological disorders and cognitive decline age related. Methylglyoxal (MG) is one of the reactive dicarbonyl precursors of AGEs, mainly generated as a by-product of glycolysis, whose accumulation induces neurotoxicity. In our study, MG cytotoxicity was evaluated employing a human stem cell-derived model, namely, neuron-like cells (hNLCs) transdifferentiated from mesenchymal stem/stromal cells, which served as a source of human based species-specific "healthy" cells. MG increased ROS production and induced the first characteristic apoptotic hallmarks already at low concentrations (≥10 µM), decreased the cell growth (≥5-10 µM) and viability (≥25 µM), altered Glo-1 and Glo-2 enzymes (≥25 µM), and markedly affected the neuronal markers MAP-2 and NSE causing their loss at low MG concentrations (≥10 µM). Morphological alterations started at 100 µM, followed by even more marked effects and cell death after few hours (5 h) from 200 µM MG addition. Substantially, most effects occurred as low as 10 µM, concentration much lower than that reported from previous observations using different in vitro cell-based models (e.g., human neuroblastoma cell lines, primary animal cells, and human iPSCs). Remarkably, this low effective concentration approaches the level range measured in biological samples of pathological subjects. The use of a suitable cellular model, that is, human primary neurons, can provide an additional valuable tool, mimicking better the physiological and biochemical properties of brain cells, in order to evaluate the mechanistic basis of molecular and cellular alterations in CNS.

Enniatin B1: Emerging Mycotoxin and Emerging Issues.

Although over the last 10 years several studies have focused on the emerging mycotoxins known as enniatins (ENNs), there is still a lack of knowledge regarding their toxicological effects and the development of a correct risk assessment. This is especially true for enniatin B1 (ENN B1), considered the younger sister of the widely studied enniatin B (ENN B). ENN B1 has been found in several food commodities and, as with other mycotoxins, presents antibacterial and antifungal properties. On the other hand, ENN B1 has shown cytotoxic activity, impairment of the cell cycle, the induction of oxidative stress, and changes in mitochondrial membrane permeabilization, as well as negative genotoxic and estrogenic effects. Overall, considering the paucity of information available regarding ENN B1, further studies are necessary to perform a risk assessment. This review summarizes information on the biological characteristics and toxicological effects of ENN B1 as well as the future challenges that this mycotoxin could present.

In Vitro Effects of Enniatin A on Steroidogenesis and Proliferation of Bovine Granulosa Cells.

The emerging Fusarium mycotoxins enniatins (ENNs) have been the focus of new research because of their well-documented existence in various cereal and grain products. Research findings indicate that reproductive disorders may be caused by exposure to Fusarium mycotoxins, but little work has evaluated ENNs on reproductive function. Therefore, to determine the effects of ENNA on the proliferation and steroidogenesis of granulosa cells (GC), experiments were conducted using bovine GC cultures. In vitro, ENNA (1−5 µM) inhibited (p < 0.05) hormone-induced GC progesterone and estradiol production. The inhibitory effect of ENNA on estradiol production was more pronounced in small- than large-follicle GC. In large-follicle GC, 0.3 µM ENNA had no effect (p > 0.10) whereas 1 and 3 µM ENNA inhibited GC proliferation. In small-follicle GC, ENNA (1−5 µM) dramatically decreased (p < 0.05) GC proliferation. Using cell number data, the IC50 of ENNA was estimated at 2 µM for both follicle sizes. We conclude that ENNA can directly inhibit ovarian function in cattle, decreasing the proliferation and steroid production of GC.

Climate Change and Effects on Molds and Mycotoxins.

Earth's climate is undergoing adverse global changes as an unequivocal result of anthropogenic activity. The occurring environmental changes are slowly shaping the balance between plant growth and related fungal diseases. Climate (temperature, available water, and light quality/quantity; as well as extreme drought, desertification, and fluctuations of humid/dry cycles) represents the most important agroecosystem factor influencing the life cycle stages of fungi and their ability to colonize crops, survive, and produce toxins. The ability of mycotoxigenic fungi to respond to Climate Change (CC) may induce a shift in their geographical distribution and in the pattern of mycotoxin occurrence. The present review examines the available evidence on the impact of CC factors on growth and mycotoxin production by the key mycotoxigenic fungi belonging to the genera Aspergillus, Penicillium, and Fusarium, which include several species producing mycotoxins of the greatest concern worldwide: aflatoxins (AFs), ochratoxins, and fumonisins (FUMs).

Replacement of animal testing by integrated approaches to testing and assessment (IATA): a call for in vivitrosi.

Alternative methods to animal use in toxicology are evolving with new advanced tools and multilevel approaches, to answer from one side to 3Rs requirements, and on the other side offering relevant and valid tests for drugs and chemicals, considering also their combination in test strategies, for a proper risk assessment.While stand-alone methods, have demonstrated to be applicable for some specific toxicological predictions with some limitations, the new strategy for the application of New Approach Methods (NAM), to solve complex toxicological endpoints is addressed by Integrated Approaches for Testing and Assessment (IATA), aka Integrated Testing Strategies (ITS) or Defined Approaches for Testing and Assessment (DA). The central challenge of evidence integration is shared with the needs of risk assessment and systematic reviews of an evidence-based Toxicology. Increasingly, machine learning (aka Artificial Intelligence, AI) lends itself to integrate diverse evidence streams.In this article, we give an overview of the state of the art of alternative methods and IATA in toxicology for regulatory use for various hazards, outlining future orientation and perspectives. We call on leveraging the synergies of integrated approaches and evidence integration from in vivo, in vitro and in silico as true in vivitrosi.

Emerging mycotoxins and reproductive effects in animals: A short review.

Emerging Fusarium mycotoxins beauvericin (BEA), enniatins (ENNs), and moniliformin (MON) are gaining increasing interest due to their wide presence especially in cereals and grain-based products. In vitro and in vivo studies indicate that Fusarium mycotoxins can be implicated in reproductive disorders in animals. Of these mycotoxins, BEA may affect reproductive functions, impairing the development of oocytes in pigs and sheep. Studies show dramatic inhibitory effects of BEA and ENNA on bovine granulosa cell steroidogenesis. ENNs also inhibit boar sperm motility and cause detrimental effects on embryos in mice and pigs. Although little data are reported on reproductive effects of MON, in vitro studies show inhibitory effects of MON on Chinese hamster ovary cells. The present review aims to summarize the reproductive toxicological effects of emerging Fusarium mycotoxins BEA, ENNs, and MON on embryo development, ovarian function, and testicular function of animals. In vitro and in vivo toxicological data are reported although additional studies are needed for proper risk assessment.

Three-dimensional spheroid cell culture of human MSC-derived neuron-like cells: New in vitro model to assess magnetite nanoparticle-induced neurotoxicity effects.

As nanoparticles (NPs) can access the brain and impact on CNS function, novel in vitro models for the evaluation of NPs-induced neurotoxicity are advocated. Three-dimensional spheroids of primary neuron-like cells (hNLCs) of human origin have been generated, from differentiation of human umbilical cord mesenchymal stem cells (MSCs). The study evaluated Fe3 O4 NP impact on the differentiation process by applying the challenge at complete 3D hNLC spheroid formation (after 4 days, T4) or at beginning of neurogenic induction/simultaneously 3D forming (T0). Different endpoints were monitored over time (up to 10 days): spheroid growth, size, morphology, ATP, cell death, neuronal markers (ß-Tub III, MAP-2, and NSE), NP uptake. At T0 application, a marked concentration- and time-dependent cell mortality occurred: effect started early (day 2) and low concentration (1 µg/ml) and exacerbated (80% mortality) after prolonged time (day 6) and increased concentrations (50 µg/ml). ATP was strikingly affected. All neuronal markers were downregulated, and spheroid morphology altered in a concentration-dependent manner (from ≥5 µg/ml) after day 2. Fe3 O4 NPs applied at complete 3D formation (T4) still induced adverse effects although less severe: cell mortality (20-60%) and ATP content decrease (10-40%) were observed in a concentration-dependent manner (from ≥ 5 µg/ml). A neuronal-specific marker effect and spheroid size reduction from 25 µg/ml without morphology alteration were evidenced. This finding provides additional information on neurotoxic effects of Fe3 O4 NPs in a new 3D hNLC spheroid model derived from MSCs that could find a consistent application as in a testing strategy serving in first step hazard identification for correct risk assessment.

Plants and zootoxins: Toxico-epidemiological investigation in domestic animals.

An epidemiological study on animal poisoning due to plants and zootoxins has been carried out by the Poison Control Centre of Milan (CAV) in collaboration with the University of Milan (Italy). During the period January 2015-March 2019, the CAV received 932 calls on animal poisonings, 12.66% (n = 118) of which were related to plants and zootoxins. Among these, 95 enquiries (80.51%) concerned exposures to plants and 23 (19.49%) to zootoxins. The dog was the species most frequently involved (67.80% of the calls, n = 80), followed by the cat (26.27%, n = 31). As for the plants, several poisoning episodes were related to glycoside-, alkaloid-, oxalate- and diterpenoid-containing species. Cycas revoluta, Euphorbia pulcherrima and Hydrangea macrophylla were the most often reported plants. The outcome has been reported for half of the episodes (51.58%, n = 49) and it was fatal for 3 animals (6.12%). Regarding the zootoxins, the majority of the enquiries were related to asp viper (Vipera aspis), but exposures to pine processionary moth (Thaumetopoea pityocampa), common toad (Bufo), fire salamander (Salamandra), and jellyfish (phylum Cnidaria) were also reported. The outcome was known in 65.22% of the cases with just one fatal episode. This epidemiological investigation depicts an interesting overview on the issue of plant and zootoxin exposures in domestic animals, highlighting the relevance of these agents as causes of animal poisoning and providing useful information for prevention and diagnosis.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro.

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.