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.

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.

In vitro copper oxide nanoparticle toxicity on intestinal barrier.

The use of CuO nanoparticles (NPs) has increased greatly and their potential effects on human health need to be investigated. Differentiated Caco-2 cells were treated from the apical (Ap) and the basolateral (Bl) compartment with different concentrations (0, 10, 50 and 100 µg/mL) of commercial or sonochemically synthesized (sono) CuO NPs. Sono NPs were prepared in ethanol (CuOe) or in water (CuOw), obtaining CuO NPs differing in size and shape. The effects on the Caco-2 cell barrier were assessed via transepithelial electrical resistance (TEER) evaluation just before and after 1, 2 and 24 hours of exposure and through the analysis of cytokine release and biomarkers of oxidative damage to proteins after 24 hours. Sono CuOe and CuOw NPs induced a TEER decrease with a dose-dependent pattern after Bl exposure. Conversely, TEER values were not affected by the Ap exposure to commercial CuO NPs and, concerning the Bl exposure, only the lowest concentration tested (10 µg/mL) caused a TEER decrease after 24 hours of exposure. An increased release of interleukin-8 was induced by sono CuO NPs after the Ap exposure to 100 µg/mL and by sono and commercial CuO after the Bl exposure to all the concentrations. No effects of commercial and sono CuO NPs on interleukin-6 (with the only exception of 100 µg/mL Bl commercial CuO) and tumor necrosis factor-α release were observed. Ap treatment with commercial and CuOw NPs was able to induce significant alterations on specific biomarkers of protein oxidative damage (protein sulfhydryl group oxidation and protein carbonylation).

In vitro evaluation of magnetite nanoparticles in human mesenchymal stem cells: comparison of different cytotoxicity assays.

This work was aimed at defining the suitable test for evaluating Fe3O4 NPs cytotoxicity after short-term exposure in human mesenchymal stem cells (hMSCs) using different viability tests, namely NRU, MTT and TB assays, paralleled by cell morphology analyses for cross checking. MTT and NRU data (culture medium with/without hMSCs plus Fe3O4NPs) indicated artificial/false increments in cell viability after Fe3O4NPs. These observations did not fit with the morphological analyses showing reduced cell density, loss of monolayer features, and morphological alterations at Fe3O4NPs ≥50 µg/ml. Fe3O4NPs alone induced a substantial increased absorbance at the wavelength required for MTT and NRU. A significant death (25%) of hMSC at Fe3O4NPs ≥10 µg/ml, with a maximum effect (45%) at 300 µg/ml after 24 h, exacerbated after 48 h, was observed when applying TB test. These results paralleled the effects on cell morphology. The optical properties and stability of Fe3O4NP suspension (tendency to agglomerate in a specific culture medium) represent factors that limit in vitro result interpretation. These findings suggest the non applicability of the spectrophotometric assays for hMSC culture conditions, while TB is an accurate method for determining cell viability after Fe3O4NP exposure in this model. In relation to NPs safety assessment: cell-based assays must be considered on case-by-case basis; selection of relevant cell models is also important for predictive toxicological studies; application of a testing strategy is fundamental for understanding the toxicity pathways driving cellular responses.

Organoids are promising tools for species-specific in vitro toxicological studies.

Organoids are three-dimensional self-aggregating structures generated from stem cells (SCs) or progenitor cells in a process that recapitulates molecular and cellular stages of early organ development. The differentiation process leads to the appearance of specialized mature cells and is connected with changes in the organoid internal structure rearrangement and self-organization. The formation of organ-specific structures in vitro with highly ordered architecture is also strongly influenced by the extracellular matrix. These features make organoids as a powerful model for in vitro toxicology. Nowadays this technology is developing very quickly. In this review we present, from a toxicological and species-specific point of view, the state of the art of organoid generation from adult SCs and pluripotent SCs: embryonic SCs or induced pluripotent SCs. The current culture organoid techniques are discussed for their main advantages, disadvantages and limitations. In the second part of the review, we concentrated on the characterization of species-specific organoids generated from tissue-specific SCs of different sources: mammary (bovine), epidermis (canine), intestinal (porcine, bovine, canine, chicken) and liver (feline, canine).

Cytotoxic and proinflammatory responses induced by ZnO nanoparticles in in vitro intestinal barrier.

ZnO nanoparticles (NPs) are widely used nowadays, thus the gastrointestinal exposure to ZnO NPs is likely to be relevant and the effects on the intestinal barrier should be investigated. Polarized Caco-2 cells were exposed from the apical (Ap) and basolateral (Bl) compartments to increasing concentrations (0, 10, 50 and 100 µg/mL) of sonochemical (sono) and commercial ZnO NPs. The transepithelial electrical resistance (TEER), cell viability, proinflammatory cytokine release and presence of protein oxidative damage were evaluated after exposure. TEER was not significantly affected by Ap exposure to either sono or commercial ZnO NPs at any tested concentrations. After Bl exposure to sono ZnO NPs (all the concentrations) and to 100 µg/mL of commercial ZnO NPs TEER was decreased (P < 0.05). Ap and Bl exposure to 100 µg/mL sono ZnO NPs and Ap exposure to 50 µg/mL commercial ZnO NPs induced a significant (P < 0.05) release of interleukin-6. A significant (P < 0.05) release of interleukin-8 was observed after Ap exposure to ZnO NPs at 100 µg/mL and after Bl exposure to sono ZnO NPs at 100 µg/mL. Ap or Bl exposure to sono or commercial ZnO NPs did not affect tumour necrosis factor-alpha secretion or protein sulphydryl oxidation. In conclusion, the ZnO NP exposure from the Ap compartment appeared almost safe, while the exposure through the basal compartment appeared to be more hazardous and the different NP size and crystallinity seem to affect the mode of action, but further studies are necessary to elucidate better these toxicity mechanisms.

Neuron-Like Cells Generated from Human Umbilical Cord Lining-Derived Mesenchymal Stem Cells as a New In Vitro Model for Neuronal Toxicity Screening: Using Magnetite Nanoparticles as an Example.

The wide employment of iron nanoparticles in environmental and occupational settings underlines their potential to enter the brain. Human cell-based systems are recommended as relevant models to reduce uncertainty and to improve prediction of human toxicity. This study aimed at demonstrating the in vitro differentiation of the human umbilical cord lining-derived-mesenchymal stem cells (hCL-MSCs) into neuron-like cells (hNLCs) and the benefit of using them as an ideal primary cell source of human origin for the neuronal toxicity of Fe3O4NPs (magnetite-nanoparticles). Neuron-like phenotype was confirmed by: live morphology; Nissl body staining; protein expression of different neuronal-specific markers (immunofluorescent staining), at different maturation stages (i.e., day-3-early and day-8-full differentiated), namely ß-tubulin III, MAP-2, enolase (NSE), glial protein, and almost no nestin and SOX-2 expression. Synaptic makers (SYN, GAP43, and PSD95) were also expressed. Fe3O4NPs determined a concentration- and time-dependent reduction of hNLCs viability (by ATP and the Trypan Blue test). Cell density decreased (20-50%) and apoptotic effects were detected at ≥10 µg/mL in both types of differentiated hNLCs. Three-day-differentiated hNLCs were more susceptible (toxicity appeared early and lasted for up to 48 h) than 8-day-differentiated cells (delayed effects). The study demonstrated that (i) hCL-MSCs easily differentiated into neuronal-like cells; (ii) the hNCLs susceptibility to Fe3O4NPs; and (iii) human primary cultures of neurons are new in vitro model for NP evaluation.

Human 3D Cultures as Models for Evaluating Magnetic Nanoparticle CNS Cytotoxicity after Short- and Repeated Long-Term Exposure.

Since nanoparticles (NPs) can translocate to the brain and impact the highly vulnerable central nervous system (CNS), novel in vitro tools for the assessment of NP-induced neurotoxicity are advocated. In this study, two types of CNS spheroids have been developed from human D384 astrocyte- and SH-SY5Y neuronal-like cells, and optimized in combination with standard assays (viability readout and cell morphology) to test neurotoxic effects caused by Fe3O4NPs, as NP-model, after short- (24­48 h; 1­100µg/ml) and long-term repeated exposure (30days; 0.1­25µg/ml). Short-term exposure of 3D-spheroids to Fe3O4NP induced cytotoxicity at 10 µg/mL in astrocytes and 25 µg/mL neurons. After long-term repeated dose regimen, spheroids showed concentration- and time-dependent cell mortality at 10 µg/mL for D384 and 0.5 µg/mL for SH-SY5Y, indicating a higher susceptibility of neurons than astrocytes. Both spheroid types displayed cell disaggregation after the first week of treatment at ≥0.1 µg/mL and becoming considerably evident at higher concentrations and over time. Recreating the 3D-spatial environment of the CNS allows cells to behave in vitro more closely to the in vivo situations, therefore providing a model that can be used as a stand-alone test or as a part of integrated testing strategies. These models could drive an improvement in the species-relevant predictivity of toxicity testing.

Cytotoxicity and proliferative capacity impairment induced on human brain cell cultures after short- and long-term exposure to magnetite nanoparticles.

Since magnetic iron oxide nanoparticles (IONP) as magnetite (Fe3 O4 NPs) have potential applications in life sciences, industrial fields and biomedical care, the risks for occupational, general population and patients rises correspondingly. Excessive IONP accumulation in central nervous system (CNS) cells can lead to a disruption of normal iron metabolism/homeostasis, which is a characteristic hallmark resembling that of several neurodegenerative disorders. Fe3 O4 NPs- versus Fe3 O4 bulk-induced toxic effects have been assessed in two human CNS cells namely astrocytes (D384) and neurons (SH-SY5Y) after short-term exposure (4-24-48 h) to 1-100 µg ml-1 , and long-term exposure to lower concentrations. Short-term Fe3 O4 NPs induced significant concentration- and time-dependent alterations of mitochondrial function in D384 (25-75% cell viability decrease): effects started at 25 µg ml-1 after 4 h, and 1 µg ml-1 after 48 h. SH-SY5Y were less susceptible: cytotoxicity occurred after 48  h only with 35-45% mortality (10-100 µg ml-1 ). Accordingly, a more marked intracellular iron accumulation was observed in astrocytes than neurons. Membrane integrity was unaltered in both CNS cell types. Lowering Fe3 O4 NP concentrations (0.05-10 µg ml-1 ) and prolonging the exposure time (up to 10 days), D384 toxicity was again observed (colony number decrease at ≥0.05 µg ml-1 , morphology alterations and colony size reduction at ≥0.5 µg ml-1 ). Effects on SH-SY5Y appeared at the highest concentration only. Fe3 O4 bulk was always remarkably toxic toward both cells. In summary, human cultured astrocytes were susceptible to both Fe3 O4 NP and bulk forms following short-term and extended exposure to low concentrations, while neurons were more resistant to NPs. Cellular iron overload may trigger adverse responses by releasing iron ions (particularly in astrocytes) thus compromising the normal functions of CNS. Copyright © 2016 John Wiley & Sons, Ltd.

Evidence for direct effects of glyphosate on ovarian function: glyphosate influences steroidogenesis and proliferation of bovine granulosa but not theca cells in vitro.

Glyphosate (GLY) is a common herbicide used worldwide but its effect on ovarian function in mammals is unknown. The aim of this study was to determine the potential endocrine disruptor effects of GLY on ovarian function evaluating cell proliferation, steroidogenesis and gene expression using bovine granulosa cells (GC) and theca cells as in vitro models. GC proliferation was impaired (P < 0.05) after exposure to GLY at 0.5, 1.7 and 5 µg ml-1 . GC progesterone production was not affected (P ≥ 0.05) at all doses tested while estradiol production was inhibited (P < 0.05) by GLY at 5 µg ml-1 . At the same concentration GLY showed no effect (P ≥ 0.05) on theca cell proliferation and steroidogenesis. At higher concentrations (0.01 and 0.3 mg ml-1 ), GLY had no significant effect (P ≥ 0.05) on GC proliferation and steroidogenesis. These studies, for the first time, suggest that GLY may affect the reproductive system in cattle via direct action on ovarian function; however, further studies will be required to understand better the mechanism of action and to determine the in vivo reproductive effects of GLY. Copyright © 2016 John Wiley & Sons, Ltd.

Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds.

Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell-cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 µM), (2) Fe3O4 nanoparticles (1-100 µg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron-astrocyte metabolic interactions.

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.

Student perspectives on the use of alternative methods for teaching in veterinary faculties.

The use of alternative methods for teaching purposes is gradually increasing in higher education. In order to evaluate the usefulness of non-animal based practical classes in veterinary science, and to inform on possible benefits and limitations of these teaching tools, a questionnaire was designed and distributed to students. Although there was no complete agreement among the student responses, it was apparent that the majority of the students would like traditional training methods to be paired with alternative approaches, and expressed their desire to be exposed to as many humane modes of learning as possible. In addition, the students agreed that alternative teaching methods for training in veterinary science can reinforce existing knowledge that is required at the clinical stage, and that they can be effective supplements to traditional training methods. It was also concluded from the study that the use of new alternative approaches is very much appreciated by the students, whereas the validity and effectiveness of these methods are debatable, suggesting that further optimisation, proper application and evaluation of these alternative methods is required.

3D human stem-cell-derived neuronal spheroids for in vitro neurotoxicity testing of methylglyoxal, highly reactive glycolysis byproduct and potent glycating agent.

Human-derived three-dimensional (3D) in vitro models are advanced human cell-based model for their complexity, relevance and application in toxicity testing. Intracellular accumulation of methylglyoxal (MGO), the most potent glycating agent in humans, mainly generated as a by-product of glycolysis, is associated with age-related diseases including neurodegenerative disorders. In our study, 3D human stem-cell-derived neuronal spheroids were set up and applied to evaluate cytotoxic effects after short-term (5 to 48 h) treatments with different MGO concentrations, including low levels, taking into consideration several biochemical endpoints. In MGO-treated neurospheroids, reduced cell growth proliferation and decreased cell viability occurred early from 5-10 µM, and their compactness diminished starting from 100 µM, apparently without affecting spheroid size. MGO markedly caused loss of the neuronal markers MAP-2 and NSE from 10-50 µM, decreased the detoxifying Glo1 enzyme from 50 µM, and activated NF-kB by nuclear translocation. The cytochemical evaluation of the 3D sections showed the presence of necrotic cells with loss of nuclei. Apoptotic cells were observed from 50 µM MGO after 48 h, and from 100 µM after 24 h. MGO (50-10 µM) also induced modifications of the cell-cell and cell-ECM interactions. These effects worsened at the higher concentrations (300-500 µM). In 3D neuronal spheroids, MGO tested concentrations comparable to human samples levels measured in MGO-associated diseases, altered neuronal key signalling endpoints relevant for the pathogenesis of neurodegenerative diseases and aging. The findings also demonstrated that the use of 3D neuronal spheroids of human origin can be useful in a strategy in vitro for testing MGO and other dicarbonyls evaluation.

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.

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.

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.

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.