In vitro effects of two environmental toxicants, beauvericin and glyphosate in Roundup, on cell numbers and steroidogenesis of bovine ovarian cells.

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.

Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs.

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.

Changes in fibroblast growth factor receptors-1c, -2c, -3c, and -4 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

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.

Species-specific models in toxicology: in vitro epithelial barriers.

Species-specific in vitro epithelial barriers represent interesting predictive tools for risk assessment evaluation in toxicological studies. Moreover, these models could be applied either as stand-alone methods for the study of absorption, bioavailability, excretion, transport, effects of xenobiotics, or through an Integrated Testing Strategy. The aim of this review is to give a comprehensive overview of in vitro species-specific epithelial barrier models from bovine, dog and swine. Bovine mammary epithelial barrier as a fundamental instrument for the evaluation of the toxicant excretion, the blood brain barrier as a useful first approach in toxicological and pharmacological studies, the porcine intestinal barrier, the canine skin barrier, and finally the pulmonary barrier from bovine and swine species are described in this review.

Fusarium mycotoxins and in vitro species-specific approach with porcine intestinal and brain in vitro barriers: A review.

Fusarium mycotoxins, such as fumonisins, trichothecenes, zearalenone and emerging fusariotoxins, common contaminants of feed and food, have received increased interest, due to the possible impact on animal and human health. In this context, it is urgent to focus our attention on fusariotoxins adverse effects, considering and analysing data in relation to their species-specificity. The in vitro approach for fusariotoxins risk assessment evaluation, through porcine epithelial barriers model, allowed to collect information on their absorption profile, bioavailability and toxicity. The aim of this review is to give an overview on Fusarium mycotoxins and their interactions with porcine intestinal and brain in vitro barriers, because they represent direct target organs of toxicity and as tools to evaluate their permeability and transport.

Changes in fibroblast growth factor 9 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n=8) or 5 to 6 (n=8) postovulation for GC and TC RNA extraction from small (1-5mm), medium (5.1-8mm), and large (8.1-18mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2)P4) follicles at both early (d 3-4) and late (d 5-6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r=-0.68), free IGF-1 (r=-0.63), and E2-to-P4 ratio (r=-0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle.

Detection of multiple mycotoxin occurrences in soy animal feed by traditional mycological identification combined with molecular species identification.

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.

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

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

Plant poisoning in domestic animals: epidemiological data from an Italian survey (2000-2011).

An Italian epidemiological study based on the human Poison Control Centre of Milan (Centro Antiveleni di Milano (CAV)) data related to domestic animal poisoning by exposure to plants, was carried out in collaboration with the Veterinary Toxicology Section of the University of Milan. It encompasses a 12-year period, from the beginning of 2000 to the end of 2011. Calls related to toxic plants accounted for 5.7 per cent of total inquiries (2150) received by CAV. The dog was the most commonly poisoned species (61.8 per cent of calls) followed by the cat (26 per cent). Little information was recorded for other species. Most exposures (73.8 per cent) resulted in mild to moderate clinical signs. The outcome was reported in only 53.7 per cent of cases, and fatal poisoning accounted for 10.6 per cent of these cases. Glycoside, alkaloid, oxalate, toxalbumin, saponin, terpene and terpenoid-containing plants were recorded and found to be responsible for intoxication. Cycas revoluta, Euphorbia pulcherrima, Hydrangea macrophylla, Nerium oleander, Rhododendron species and Prunus species were the plants most frequently involved. Epidemiological data from this Italian survey provide useful information on animal exposure to plants and confirm the importance of plants as causative agents of animal poisoning.

Animal poisoning in Italy: 10 years of epidemiological data from the Poison Control Centre of Milan.

From 2000 to 2010, the Poison Control Centre of Milan (CAV), in collaboration with the University of Milan, Faculty of Veterinary Medicine, Department of Veterinary Sciences and Technologies for Food Safety, Toxicology Section, collected epidemiological information related to animal poisoning and classified it in an organised and computerised data bank. Data recorded were predominantly related to small animals and to some extent to horses, ruminants and other food-production animals. Few calls were registered involving exotics and no information was recorded on wildlife. The dog was reported to be the most common species involved in animal poisoning, and pesticides constituted the primary group of toxicants. In the case of pets, 'drugs' including veterinary parasiticide and drugs for human use constituted the second class of toxicants responsible for poisoning followed by household products, plants, zootoxins and metals. With regard to horses and farm animals, the second group consisted of phytotoxins, even if only episodically. In Italy, published data on this subject are scarce but this information is crucial for better management of the poisoning of domestic animals in an effort to reduce mortality.

Microarray analysis of insulin-like growth factor-I-induced changes in messenger ribonucleic acid expression in cultured porcine granulosa cells: possible role of insulin-like growth factor-I in angiogenesis.

Insulin-like growth factor-I in conjunction with gonadotropins are important stimulators of mitosis and ovarian steroid production by granulosa and thecal cells, which are required for normal oocyte development and hormonal feedback signaling to the hypothalamus and pituitary. However, a comprehensive evaluation of the changes in gene expression induced by IGF-I has not been conducted. Our objective was to characterize granulosa cell gene expression in response to IGF-I treatment. Porcine granulosa cells were pooled in 4 biological replicates and treated with FSH (baseline) or FSH+IGF-I for 24 h in vitro. The RNA was collected and hybridized to 8 Affymetrix Porcine GeneChips (Affymetrix, Santa Clara, CA) in a paired design. Differentially regulated gene sequence element sets (P < 0.01) were used as queries in the UniGene database searching for annotated genes. Abundance of messenger RNA (mRNA) for genes differentially expressed in the microarray analysis was determined through multiplex assays of one-step real-time reverse transcription-PCR and further analyzed under a statistical model including the fixed effect of treatment. A total of 388 gene sequence element sets were differentially expressed, and 42 matched annotated genes in the UniGene database. Of the 3 upregulated target genes selected for further quantitative reverse transcription-PCR analysis, only FGF receptor 2 III c (FGFR2IIIc) mRNA abundance was significantly increased by IGF-I. Of the 3 downregulated target genes selected for further analysis, only thrombospondin-1 (THBS1) mRNA abundance was significantly decreased by IGF-I. Further study revealed that neither FSH nor estradiol affected the IGF-I-induced suppression of THBS1 mRNA abundance. These results provide the first comprehensive assessment of IGF-I-induced gene expression in granulosa cells and will contribute to a better understanding of the molecular mechanisms of IGF-I regulation of follicular development. Involvement of FGFR2IIIc and THBS1 in mediating IGF-I-induced granulosa cell steroidogenesis and proliferation during follicular development is novel, but their specific roles will require further elucidation.

Aflatoxin M1 absorption and cytotoxicity on human intestinal in vitro model.

Aflatoxin M1 (AFM1) is the principal hydroxylated Aflatoxin B1 (AFB1) metabolite and is detected in milk of mammals, after consumption of feed contaminated with AFB1. As it is classified as probable human carcinogen (group 2B of the IARC), most countries have regulated its maximum allowed levels in milk in order to reduce AFM1 risk (50 ng/kg the EU and 500 ng/kg in the USA). It was demonstrated that if AFB1 must be converted into its reactive epoxide to exert its effects, and the protein binding may play an important role in its cytotoxicity. Conversely, the AFM1 epoxidation in human liver microsomes is very limited and studies with human cell line (MCL5), expressing or not expressing cytochrome P450 enzymes, demonstrated a direct toxic potential of AFM1 in absence of metabolic activation. For this reason, while AFM1 is generally considered a detoxification product of AFB1 relatively to carcinogenicity and mutagenicity property, this is not always true for cytotoxicity activity. Aim of this work is to evaluate the intestinal absorption of AFM1 using a human in vitro model, the Caco-2 cell line. Either the parental Caco-2 cell line or its derived clone TC7, with higher metabolic competence, have been used. They were treated with different concentrations of AFM1, that mirror the milk contamination level (0.3-32 nM corresponding to 10-10,000 ng/kg), either in undifferentiated or in differentiated phase of growth. After 48 h of treatment in serum free medium, a dose dependent absorption of AFM1 has been detected in both cell lines, especially in differentiated cells, while, no appreciable effects on cell viability were observed, except for a general cellular suffering, revealed by LDH release, particularly evident in the undifferentiated cells. As well, no metabolites or AFM1 conjugates have been detected. The present results may be crucial for the evaluation of human risk to AFM1 exposure, in particular for children's population, due to their large use of milk and derivatives.

Aflatoxin B1 binding to sorbents in bovine ruminal fluid.

A recent approach to the problem of contamination of agricultural products by aflatoxin B(1) (AFB(1)) is to add non-nutritional adsorbents to animal diets in order to sequester ingested aflatoxins. We conducted in vitro experiments to develop a rapid and cheap model using ruminal fluid to assess the ability of sorbent materials to bind AFB(1). Seven sorbents (hydrated sodium calcium aluminosilicate; clinoptilolite; zeolite; two types of bentonite; sepiolite; and PHIL 75), commonly added to bovine diets were incubated in water and ruminal fluid in the presence of AFB(1). Hydrated sodium calcium aluminosilicate, sepiolite and one of the bentonites bound 100% of the AFB(1) in the presence of both ruminal fluid and water; clinoptilolite bound about 80% of AFB(1) in both liquids; whereas the affinities for the mycotoxin of zeolite (50%) and the other sample of bentonite (60%) in water seem to be increased by about 40% in ruminal fluid incubations. PHIL 75 had the poorest binding ability: about 30% in water and 45% in ruminal fluid. In view of the differences in toxin binding in water and ruminal fluid, it is preferable to use the ruminal fluid model for the in vitro pre-screening of sorbent materials potentially useful as adjuvants to ruminant feeds.

Absorption of fumonisin B1 and aminopentol on an in vitro model of intestinal epithelium; the role of P-glycoprotein.

The aim of the present paper is to evaluate the absorption of fumonisin B1 and its principal metabolite, aminopentol on a human intestinal model, Caco-2 cells, cultured on semi-permeable inserts, that reproduces the two different intestinal compartments: luminal (apical) and serosal (basolateral) side. Following separate exposure in apical and in basolateral compartments, aminopentol passage through the cell layer (in particular from basolateral to apical direction) was shown, while it was not observed for the parent compound. The different aminopentol distribution between the two compartments of the culture system, and its variation in presence of verapamil or probenecid (P-gp and MRP inhibitors respectively), strongly suggests the involvement of P-glycoprotein in the influx/efflux mechanisms of aminopentol in the intestinal cells, reducing its oral bioavailability.

Evaluation of Fumonisin B(1) and its metabolites absorption and toxicity on intestinal cells line Caco-2.

The aim of the present paper is to investigate intestinal absorption and toxicity of Fumonisin B(1) (FB(1)) and its partially (PHFB(1) and PHFB(2)) and totally hydrolyzed (HFB(1)) metabolites, using the human intestinal cell line Caco-2, a very well known in vitro model of intestinal epithelium for absorption and metabolism studies. Caco-2 cells were treated for 48 h with several toxin concentrations (in the range of 1-138 microM). At the end of exposure period, no significant variation on cell viability has been observed with all chemicals tested, either in undifferentiated cells or in differentiated ones, suggesting a poor toxicity of these mycotoxins for intestinal cells. In any case, FB(1) appears the most active in this respect. For which concerns the cellular absorption, FB(1), PHFB(1) and PHFB(2) are never detected into Caco-2 cells. On the contrary, a dose-dependent absorption of HFB(1) has been observed in differentiated cells, which express enzymatic and metabolic characteristics of mature enterocytes. Thus HFB(1), losing the tricarballylic acid chain, is more bioavailable than FB(1) on intestinal cell, supporting the hypothesis that in risk evaluation of fumonisins exposure its metabolites are also relevant.

Fumonisin B1 metabolism by bovine liver microsomes.

Only limited and contrasting information is available about the metabolic fate in cattle of fumonisin B1, a mycotoxin produced by moulds of Fusarium. This study was carried out to evaluate the hepatic metabolism of fumonisin B1 by bovine liver microsomes. No biodegradation or metabolization of the mycotoxin by liver microsomes was detectable after incubating fumonisin B1 with bovine microsomes in the presence of a regenerating system for 1 h. No aminopolyol 1, aminopolyol 2 or aminopentol, metabolites of fumonisin B1, were detected in any of the incubated samples. The tolerance of ruminants to fumonisin B1 is apparently not dependent on its detoxification in the rumen.