AhR-activating pesticides increase the bovine ABCG2 efflux activity in MDCKII-bABCG2 cells

In bovine mammary glands, the ABCG2 transporter actively secretes xenobiotics into dairy milk. This can have significant implications when cattle are exposed to pesticide residues in feed. Recent studies indicate that the fungicide prochloraz activates the aryl hydrocarbon receptor (AhR) pathway, increasing bovine ABCG2 (bABCG2) gene expression and efflux activity. This could enhance the accumulation of bABCG2 substrates in dairy milk, impacting pesticide risk assessment. We therefore investigated whether 13 commonly used pesticides in Europe are inducers of AhR and bABCG2 activity. MDCKII cells expressing mammary bABCG2 were incubated with pesticides for up to 72 h. To reflect an in vivo situation, applied pesticide concentrations corresponded to the maximum residue levels (MRLs) permitted in bovine fat or muscle. AhR activation was ascertained through CYP1A mRNA expression and enzyme activity, measured by qPCR and 7-ethoxyresorufin-\u39f-deethylase (EROD) assay, respectively. Pesticide-mediated increase of bABCG2 efflux activity was assessed using the Hoechst 33342 accumulation assay. For all assays, the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. At 10-fold MRL concentrations, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole significantly increased CYP1A1 mRNA levels, CYP1A activity, and bABCG2 efflux activity compared to the vehicle control. In contrast, dimethoate, dimethomorph, glyphosate, iprodione, methiocarb and thiacloprid had no impact on AhR-mediated CYP1A1 mRNA levels, CYP1A activity or bABCG2 efflux. In conclusion, the MDCKII-bABCG2 cell model proved an appropriate tool for identifying AhR- and bABCG2-inducing pesticides. This provides an in vitro approach that could reduce the number of animals required in pesticide approval studies

Conformity and controversies in the diagnosis, staging and follow-up evaluation of canine nodal lymphoma: a systematic review of the last 15 years of published literature

Diagnostic methods used in the initial and post-treatment evaluation of canine lymphoma are heterogeneous and can vary within countries and institutions. Accurate reporting of clinical stage and response assessment is crucial in determining the treatment efficacy and predicting prognosis. This study comprises a systematic review of all available canine multicentric lymphoma studies published over 15 years. Data concerning diagnosis, clinical stage evaluation and response assessment procedures were extracted and compared. Sixty-three studies met the eligibility criteria. Fifty-five (87.3%) studies were non-randomized prospective or retrospective studies. The survey results also expose variations in diagnostic criteria and treatment response assessment in canine multicentric lymphoma. Variations in staging procedures performed and recorded led to an unquantifiable heterogeneity among patients in and between studies, making it difficult to compare treatment efficacies. Awareness of this inconsistency of procedure and reporting may help in the design of future clinical trials

RNA sequencing-based whole-transcriptome analysis of friesian cattle fed with grape pomace-supplemented diet

Grape pomace (GPO), the main by-product of the wine making process, is a rich source of polyphenols with potent antioxidant properties. Recently, GPO has emerged as a potential feed additive in livestock nutrition, with several reports describing its beneficial effects on animals’ overall health status or production traits. However, little is known about it from a molecular biology standpoint. In the present study, we report the first RNA sequencing-based whole-transcriptome profiling of Friesian calves fed with a GPO-supplemented diet. We identified 367 differentially expressed genes (p < 0.05) in the GPO-supplemented calves (n = 5), when compared with unsupplemented control group (n = 5). The pathway analysis showed that ‘cholesterol lipid biosynthesis’ was the most negatively-enriched (p < 0.001) pathway in the GPO-supplemented animals. In specific terms, five important genes coding for cholesterol biosynthesis enzymes, namely the Farnesyl-diphosphate Farnesyltransferase 1 (FDFT-1), Squalene Epoxidase (SQLE), NAD(P)-dependent Steroid Dehydrogenase-like (NSDHL), Methylsterol Monooxygenase (MSMO)-1, and Sterol-C5-desaturase (SC5D), two major transcription factors (the Sterol Regulatory Element-binding Transcription Factor 1 and 2), as well as the Low-Density Lipoprotein Receptor (LDLR), were all downregulated following GPO supplementation. Such an effect was mirrored by a reduction of blood cholesterol levels (p = 0.07) and a lowered (p < 0.001) Malondialdehyde (lipid oxidation marker) level in carcasses. We provide evidence on the effects of GPO-supplemented diets on the whole-transcriptome signature in veal calves, which mainly reflects an antioxidant activity

The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study

Background: Canine lymphoma represents the most frequent haematopoietic cancer and it shares some similarities with human non-Hodgkin lymphoma. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during invasion and proliferation of malignant cells; however, little is known about their role in canine haematologic malignancies. The aim of this study was to investigate the mRNA and protein expression of VEGF and the most relevant MMPs in canine lymphoma. Lymph node aspirates from 26 B-cell and 21 T-cell lymphomas were collected. The protein expression levels of MMP-9, MMP-2 and VEGF-A were evaluated by immunocytochemistry, and the mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164 were measured using quantitative RT-PCR.Results: MT1-MMP, TIMP-1 and RECK mRNA levels were significantly higher in T-cell lymphomas than in B-cell lymphomas. Higher mRNA and protein levels of MMP-9 and VEGF-A were observed in T-cell lymphomas than in B-cell lymphomas and healthy control lymph nodes. A positive correlation was found between MMP-9 and VEGF-A in T-cell lymphomas. Moreover, MMP-9, MT1-MMP, TIMP-1 and VEGF-A were expressed at the highest levels in high-grade T-cell lymphomas.Conclusions: This study provides new information on the expression of different MMPs and VEGF in canine lymphoma, suggesting a possible correlation between different MMPs and VEGF, immunophenotype and prognosis

Matrix metalloproteinases and their inhibitors in canine mammary tumors

BACKGROUND: Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. RESULTS: MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. CONCLUSIONS: Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an \u201cin house\u201d library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT\u2013dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., \u3b1155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations

Contribution of \u2013omics methodologies to veterinary pharmacology and toxicology, with special emphasis on drug metabolizing enzymes and drug transporters

Drug metabolizing enzymes (DMEs) and drug transporters (DTs) play a crucial role in determining the kinetics and, eventually, the mechanism of action of drugs, toxins and anthropogenic compounds. Molecular studies about the expression, regulation and biological activity of DMEs/DTs are historically based on the dogma of molecular biology (DNA-mRNA-protein). However, this process is a more complex event. Different mechanisms of regulation contribute to DMEs/DTs gene transcription and translation; moreover, posttranscriptional and posttranslational events may impact on gene expression and ultimately on the amino acid sequence of coded protein (1). In the past two decades, a revolution in the scientific approach to life sciences occurred; researchers moved from studying single genes, mRNAs, proteins or metabolites to studies encompassing entire genomes, transcriptomes, proteomes, and metabolomes. These approaches enabled pharmaco-toxicologists to find new drug targets, identify biomarkers of effect/exposure and unraveling mechanistic relationships (2-4). However, some issues still remain unsolved. Interindividual differences in DMEs/DTs expression and biological activity (pharmacogenetics), as well as other factors like epigenetics, long non-coding RNAs, and gut microbiota, may result in altered drug metabolism and, hence, variations in efficacy, toxicity and adverse reactions (5-7). The aforementioned high-throughput methodologies have also been increasingly used in veterinary sciences, e.g. to identify biomarkers of illicit growth promoters misuse (8), in nutrigenomics (9), in mycotoxins mechanistic toxicology (10), in animal genetics (11). Moreover, an increasing number of papers in which DMEs/DTs were either specifically than indirectly subject of investigation have also been published, including those aiming to identify biomarkers, find non-canonical regulatory pathways, estimate the consequences of altered gene expression, and discover pathways beyond drug metabolism involved in mechanistic pharmaco-toxicology (12-18). In conclusion, omics tools can accelerate our understanding of gene/protein/metabolite networks resulting from the exposure of veterinary species to xenobiotics. Likewise to humans, these techniques may contribute to a better understanding of interindividual and species-differences in DMEs/DTs transcriptional regulation and, consequently, in kinetics, susceptibility and response to xenobiotics. References (1) Glubb and Innocenti. Wiley Interdiscip Rev Syst Biol Med 2011; 3: 299-313. (2) Kaddurah-Daouk et al. Clin Pharmacol Ther 2015; 98: 71-75. (3) Dellafiora and Dall\u2019Asta. Toxins 2017; 9: 18. (4) Joseph et al. J Appl Toxicol 2013; 33: 1193\u20131202. (5) Ahmed et al. Genomics Proteomics Bioinformatics 2016;14: 298-313. (6) Huang et al. J Appl Toxicol 2018; doi: 10.1002/jat.3595. (7) Yu et al. Acta Pharm Sin B 2017; 7:241-248. (8) Riedmaier et al. Anal Chem 2012; 84: 6863-6868. (9) Osorio et al. Small Ruminant Res 2017; 154: 29-44. (10) Wang et al. Mol Cell Proteomics 2011; 10: M111.008748. (11) Wickramasinghe et al. Livest Sci 2014; 166: 206-216. (12) Craft et al. J Vet Intern Med 2017; 31:1833\u20131840. (13) Giantin et al. Vet J 2016; 212: 36-43. (14) Heikkinen et al. Pharm Res 2015; 32: 74\u201390. (15) Howard et al. Sci Rep 2017; 7: 1357. (16) Li et al. In Vitro Cell Dev Biol - Animal 2017; 53: 293-303. (17) Reddy et al. Asian-Australas J Anim Sci 2018; 31: 138-148. (18) Visser et al. J Vet Pharmacol Ther 2017; 40: 583-590

Efficacy of chlortetracycline for controlling goat coccidiosis in Burundi.

Eighteen cross-bred goats of Burundi naturally infected to varying degrees with multiple coccidia species of the genus Eimeria were orally administered 25 mg/kg body weight/day chlortetracycline. Effectiveness percentages more elevated than 99.0% were reached within the 9th day of treatment. No adverse reactions have ever been reported. Results demonstrate that the antibiotic is effective for the control of coccidiosis of goats naturally infected in Burundi

Efficacy of ivermectin in reducing gastrointestinal nematode fecal egg counts in goats in Burundi.

Cross-bred goats in Burundi infested with gastrointestinal nematodes were submitted to fecal investigations and injected subcutaneously with ivermectin. In Experiment 1, goats were treated with 200 mu g kg(-1) bw ivermectin. In Experiment 2, animals were administered twice that dose. In Experiment 3, goats suspected to be resistant to other anthelmintics were treated with 200 mu g kg(-1) bw ivermectin. In Experiment 4, two doses of the same strength were injected with an interval of 7 days. Results demonstrate that 200 mu g kg(-1) bw ivermectin is effective for the control of gastrointestinal nematodes of goats in Burundi; this dosage is also effective against nematodes suspected to be resistant to other anthelmintics. The administration of 400 mu g kg(-1) bw did not induce greater or more prolonged effectiveness percentages. The supposed decrease of ivermectin's residual activity on Day 28 might be avoided by administering two doses with an interval of 7 days. No adverse effects were observed in treated animal