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Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Soy products are a main component of animal feed. Because mycotoxins may harm farm animals, undermining productivity and health, a mycological and toxigenic screening was carried out on 36 batches used in animal feed, collected in 2008, 2009 and 2010 in Italy. The investigated mycoflora of a subset of soy seed (n = 6) suggested that Aspergillus spp. and Fusarium spp. frequently colonize soy seeds. Aflatoxins, fumonisins and deoxynivalenol were detected in 88.9%, 72.2% and 30.6% of samples, respectively. Co-occurrence of at least two toxins was observed in 72% of cases. The molecular analysis of the Fusarium spp. population identified Fusarium verticillioides as potential producers of fumonisins, but no known deoxynivalenol producers were detected. It is suggested that the widespread presence of toxins can be due to non-optimal storing conditions of the feed. Moreover, our results suggest that mycotoxin thresholds should be adapted to consider the frequent case of toxin co-occurrence. This approach would better reflect the real toxigenic risk of feedstuffs.

The inherent adaptability of human mesenchymal stromal cells (hMSCs) to differentiate into neural lineages provides a valuable resource for investigating potential neurotoxicity in humans. By harnessing the ability of hMSCs to transform into astrocytes, we can evaluate the effects of various agents on these vital cells. Our protocol employs hMSCs sourced from umbilical cord tissue, ensuring a readily available supply of high-quality cells. The hMSC-to-neural workflow encompasses six essential steps: hMSC culture, followed by the generation of embryoid bodies (EBs) from these cells on specialized surfaces. Next, EBs and cells are expanded in a growth-promoting medium, directing them toward neural lineages. Subsequent differentiation into immature astrocytes is achieved through the use of specific factors. The process continues with the maturation of EBs/cells into astrocyte-like cells (hALCs) under optimized conditions, culminating in the final development of hALCs in a specialized medium. This methodology yields cells that display astrocyte morphology and express characteristic markers such as GFAP and S100β. The protocol is efficient, requiring roughly 6 weeks to generate hALCs from primary hMSCs without genetic manipulation. The application of hMSCs in evaluating cell damage triggered by neurotoxicants like MeHg and MGO underscores their potential as a valuable component within a more extensive battery of neurotoxicity tests.

Copper is an essential trace element but becomes toxic in overexposed animals. Sheep are the domestic species most prone to chronic copper poisoning, as a slight increase in the dietary concentration can lead to liver accumulation and the development of clinical signs in this species. Common sources of copper in the diet are feed additives and mineral supplements, which are commonly used in pigs and poultry. Recently, new copper supplements were registered for animal nutrition, including copper bilysinate. This study describes an episode of presumed chronic copper poisoning in Five Texel sheep, which were exposed to a compound feed containing copper bilysinate. Four weeks after the introduction of the compound feed into the diet, the first animal started to show typical clinical signs of chronic copper poisoning and died, followed by another animal a week later. Despite removing the compound feed from the diet, a third sheep died 3 weeks later. Two animals survived and fully recovered. Necropsy and histology showed characteristic gross and microscopical lesions typical of copper poisoning. The case report highlights the potential toxic effect of copper bilysinate in sheep.

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Beauvericin is an emerging Fusariotoxin naturally occurring in cereal grains throughout the world whereas glyphosate (N-phosphonomethyl-glycine) is a non-selective systemic herbicide used worldwide. The purpose of this study is to evaluate a newly developed ovarian cell culture system (that includes both granulosa and theca cells) as an in vitro model for toxicological studies. Specifically, the effects of beauvericin and glyphosate in formulation with Roundup on ovarian cell numbers and steroid production were evaluated. Ovaries collected from cattle without luteal structures were sliced into 30-70 pieces each, and granulosa and theca cells were collected. Harvested cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 48 h in serum-free medium containing testosterone (500 ng/mL; as an estrogen precursor) with the following eight treatments: (1) controls, (2) FSH (30 ng/mL) alone, (3) FSH plus insulin-like growth factor-1 (IGF1; 30 ng/mL), (4) FSH plus IGF1 plus beauvericin (3 µM), (5) FSH plus IGF1 plus glyphosate in Roundup (10 µg/mL), (6) FSH plus IGF1 plus fibroblast growth factor 9 (FGF9, 30 ng/mL), (7) a negative control without added testosterone, and (8) IGF1 plus LH (30 ng/mL) with basal medium without added testosterone. In the presence of FSH, IGF1 significantly increased cell numbers, estradiol and progesterone production by severalfold. Glyphosate in Roundup formulation significantly inhibited IGF1-induced cell numbers and estradiol and progesterone production by 89-94%. Beauvericin inhibited IGF1-induced cell numbers and estradiol and progesterone by 50-97% production. LH plus IGF1 significantly increased androstenedione secretion compared with controls without added testosterone indicating the presence of theca cells. In conclusion, the present study demonstrates that toxicological effects of beauvericin and glyphosate in Roundup formulation are observed in a newly developed ovarian cell model system and further confirms that both glyphosate and beauvericin may have the potential to impair reproductive function in cattle., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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 β-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., (© 2023. Controlled Release Society.)

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.

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., (© 2023 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

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.

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 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., (© 2022 John Wiley & Sons, Ltd.)

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.

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O 2 ) and low (5% O 2 ) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Fe 3 O 4 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 (β-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. Fe 3 O 4 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 Fe 3 O 4 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., (© 2022 John Wiley & Sons, Ltd.)

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., (© 2022. The Author(s).)

The various fibroblast growth factors (FGF) regulate their function via binding to 4 main FGF receptor (FGFR) subtypes and their splice variants, FGFR1b, FGF1c, FGFR2b, FGFR2c and FGFR3c and FGFR4, but which of these FGFR are expressed in the granulosa (GC) and theca cells (TC), the 2 main cell layers of ovarian follicles, or change during follicular development is unknown. We hypothesized that FGFR1c, FGFR2c and FGFR3c (but not FGFR4) gene expression in GC (but not TC) would change with follicular development. Hence, the objective of this study was to determine if abundance of FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in Holstein cattle exhibiting regular estrous cycles. Estrous cycles of non-lactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or d 5 to 6 (n = 8) post-ovulation for GC and TC RNA extraction from small (1-5 mm), medium (5.1 to 8 mm) or large (8.1-18 mm) follicles for real-time PCR analysis. In GC, FGFR1c and FGFR2c mRNA relative abundance was greater in estrogen (E2)-inactive (ie, concentrations of E2 < progesterone, P4) follicles of all sizes than in GC from large E2-active follicles (ie, E2 > P4), whereas FGFR3c and FGFR4 mRNA abundance did not significantly differ among follicle types or days post-estrus. In TC, medium E2-inactive follicles had greater FGFR1c and FGFR4 mRNA abundance than large E2-active and E2-inactive follicles on d 5 to 6 post-ovulation whereas FGFR2c and FGFR3c mRNA abundance did not significantly differ among follicle types or day post-estrus. In vitro experiments revealed that androstenedione increased abundance of FGFR1c, FGFR2c and FGFR4 mRNA in GC whereas estradiol decreased FGFR2c mRNA abundance. Neither androstenedione nor estradiol affected abundance of the various FGFR mRNAs in cultured TC. Taken together, the findings that FGFR1c and FGFR2c mRNA abundance was less in GC of E2-active follicles and FGFR1c and FGFR4 mRNA was greater in TC of medium inactive follicles at late than at early growing phase of the first dominant follicle support an anti-differentiation role for FGF and their FGFR as well as support the idea that steroid-induced changes in FGF and their receptors may regulate selection of dominant follicles in cattle., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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).

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., Competing Interests: Declaration of competing interest The authors have declared no potential conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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)., (© 2020 John Wiley & Sons, Ltd.)

Background: There is extensive literature on animal poisoning from plants and mushrooms worldwide; however, there is limited account of poisoning from South Africa., Methods: This study sought to describe and provide an overview of animal poison exposures in South Africa. Poisoning episodes reported to the Poisons Information Helpline of the Western Cape, jointly run by the Poisons Information Centres at the Red Cross War Memorial Children's Hospital and Tygerberg Hospital over a period of approximately 2.5 years, from June 2015 to November 2017, were analysed to identify exposure patterns, severity and clinical outcomes., Results: Alien plant species accounted for most cases (n=10) of reported poison exposures. Among the 26 recorded animal poisoning episodes, the dog was the most commonly implicated species (n=24), whereas just two enquiries were related to other animals (one rabbit and one cow). There were 20 plant cases and 6 mushroom cases (all dogs). There was only one fatal case involving cycad in a dog., Conclusion: Features of animal poisoning in South Africa were similar to those in other countries. The reported cases of animals exposed to poisonous plants and mushrooms could represent only a fraction of the actual exposures. Since most reported cases involved taxa that could not be identified to species level, more attention should be paid in case reporting and in animal poisoning prevention, engaging the public to enable people to recognise potentially hazardous plants and reduce the risk of poisoning in animals., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2020. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ.)

Animal poisoning by chemicals (pesticides and household products) and drugs is a frequent occurrence and special attention should be paid to this phenomenon to improve prevention and treatment strategies and because of the fundamental role that animals may play as bioindicators. From January 2017 to March 2019 the Poison Control Centre of Milan (CAV) in collaboration with the University of Milan, collected and analyzed epidemiological data on animal poisoning. During this period, the CAV received a total of 442 enquiries on domestic animal poisoning episodes and, among these, 80.3 % were related to chemicals and drugs. Pesticides and drugs were the two major causes of poisoning (34.1 % and 33.5 %, respectively), followed by household products (29.3 %) and other causative agents (3.1 %, n = 11). In conclusion, these findings can provide useful information for the identification and monitoring of known and emerging toxicants, with positive repercussions on human, animal and environmental health., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Food and feed contamination by emerging mycotoxins beauvericin and enniatins is a worldwide health problem and a matter of great concern nowadays, and data on their toxicological behavior are still scarce. As ingestion is the major route of exposure to mycotoxins in food and feed, the gastrointestinal tract represents the first barrier encountered by these natural contaminants and the first structure that could be affected by their potential detrimental effects. In order to perform a complete and reliable toxicological evaluation, this fundamental site cannot be disregarded. Several in vitro intestinal models able to recreate the different traits of the intestinal environment have been applied to investigate the various aspects related to the intestinal toxicity of emerging mycotoxins. This review aims to depict an overall and comprehensive representation of the in vitro intestinal effects of beauvericin and enniatins in humans from a species-specific perspective. Moreover, information on the occurrence in food and feed and notions on the regulatory aspects will be provided.

Episodes of indoor exposure to toxic compounds in companion animals are frequent. Toxico-epidemiological data on this matter are fundamental not only to take proper corrective actions but especially because domestic animals represent crucial sentinels for environmental toxicants. In this review we will present an overview on indoor poisoning of companion animals by chemicals in Europe, providing information on trends and emphasizing emerging indoor contaminants. In general, pesticides are the most frequent class of toxicants involved in indoor animal poisoning episodes in Europe. Among them, insecticides (in particular anticholinesterase compounds) and anticoagulant rodenticides are frequently implicated, but molluscicides (i.e. metaldehyde) together with household products have also shown a growing or at least stable trend in the poisoning episodes. Considering the emerging molecules, neonicotinoids (imidacloprid and acetamiprid) and glyphosate formulations have been pointed out, whereas with regard to households, besides the well-known ethylene glycol, attention should be paid to emerging products such as sodium polyacrylate polymeric hydrogels, xylitol, glowsticks and liquid laundry detergent capsules. The environmental contamination by chemical households is a global challenge and bioindicators like pets may be valuable tools to perform a comprehensive monitoring and risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Indoor plant poisoning poses serious threats to companion animals. One of the major reasons of this kind of hazard can be identified in the increased amount of time that the pets spend indoor, sharing the domestic environment with their owners. In this review, the toxic houseplants most commonly associated with companion animal poisoning in Europe and well-documented in the literature are emphasized. An analysis of the major and emerging plant species accountable for companion animal poisoning is proposed, in order to provide a framework of the factors influencing these incidents. Indeed, knowing the way substances may induce toxic effects in companion animals can be useful in allowing easier diagnosis and treatment processes. In conclusion, the Authors argue that a better characterization of the phenomenon, as well as of its extent, would be allowed by the availability of a centralized system for the data collection. Furthermore, better information and awareness on the issue may help developing a focused corrective approach to prevent indoor pet poisoning in Europe., (Copyright © 2020 Bertero, Fossati and Caloni.)

Beauvericin (BEA) is a natural bioactive compound, with a dual nature. On the one hand, the peculiar characteristics of its molecule confer to BEA interesting properties, such as antibacterial, antiviral, antifungal, antiparasitic, insecticidal and anticarcinogenic activities. On the other hand, it is a natural contaminant of food and feed commodities, and an emerging mycotoxin, but lacks a toxicological risk assessment evaluation for long term exposure. This review aims to provide a global and comprehensive overview on BEA from its biological activities, to its in vivo and in vitro toxicological effects covering the multifaceted nature of this substance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Data obtained from samples of poisoned domestic and wild animals sent for toxicological evaluation during the period between 2005 and 2014 have been analyzed. Among the 4606 tested samples, the majority of which were collected in the northern regions of Italy, 2006 (43.55%) were found positive for pesticides. Analytical detections were performed via solvent extraction followed by separation and chromatographic characterization and all the methods applied for the toxicological investigations were developed by IZSLER. Insecticides, mainly represented by acetylcholinesterase inhibitors (carbamates 17.55%, n = 352; organophosphates 15.15%, n = 304) and organochlorines (29.21%, n = 586), were found to be the first category of pesticides involved in intoxications, in both domestic and in wild animals, followed by rodenticides (anticoagulant rodenticides 21.09%, n = 423; zinc phosphide 2.59%, n = 52; chloralose 0.95%, n = 19 and thallium 0.15%, n = 3) and molluscicides (metaldehyde 6.63%, n = 133). Second and third generation anticoagulants (bromadiolone and brodifacoum) were the most represented (10.52%, n = 211) but also first generation compounds (i.e. coumatetralyl and warfarin) were still found responsible of intoxications. Even if some pesticides are frequently involved in domestic animal poisoning (i.e. metaldehyde and strychnine), they did not show the same diffusion in wild animals. In particular, unlike domestic species, cyanide and pyrethroids have not been found responsible of intoxications in wild animals. Interestingly, a great number of positive samples involved banned pesticides like α- (14.41%, n = 289) and β- (14.16%, n = 284) endosulfan, carbofuran (5.73%, n = 115), methamidophos (9.47%, n = 190), strychnine (6.23%, n = 125) but, on the other hand, many positives were due to the exposure to commercially available products (i.e. metaldehyde and anticoagulant rodenticides). Thus, together with measures aimed to reduce illegal uses, educational campaigns and a wider range of compounds to detect would be beneficial in order to address the issue of animal poisoning, which besides has also repercussions on environmental and public health., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This work was aimed at defining the suitable test for evaluating Fe 3 O 4 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 Fe 3 O 4 NPs) indicated artificial/false increments in cell viability after Fe 3 O 4 NPs. These observations did not fit with the morphological analyses showing reduced cell density, loss of monolayer features, and morphological alterations at Fe 3 O 4 NPs ≥50 μg/ml. Fe 3 O 4 NPs alone induced a substantial increased absorbance at the wavelength required for MTT and NRU. A significant death (25%) of hMSC at Fe 3 O 4 NPs ≥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 Fe 3 O 4 NP 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 Fe 3 O 4 NP 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.