New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines.

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.

Generation and characterization of cytochrome P450 3A74 CRISPR/Cas9 knockout bovine foetal hepatocyte cell line (BFH12).

In human, the cytochrome P450 3A (CYP3A) subfamily of drug-metabolizing enzymes (DMEs) is responsible for a significant number of phase I reactions, with the CYP3A4 isoform superintending the hepatic and intestinal metabolism of diverse endobiotic and xenobiotic compounds. The CYP3A4-dependent bioactivation of chemicals may result in hepatotoxicity and trigger carcinogenesis. In cattle, four CYP3A genes (CYP3A74, CYP3A76, CYP3A28 and CYP3A24) have been identified. Despite cattle being daily exposed to xenobiotics (e.g., mycotoxins, food additives, drugs and pesticides), the existing knowledge about the contribution of CYP3A in bovine hepatic metabolism is still incomplete. Nowadays, CRISPR/Cas9 mediated knockout (KO) is a valuable method to generate in vivo and in vitro models for studying the metabolism of xenobiotics. In the present study, we successfully performed CRISPR/Cas9-mediated KO of bovine CYP3A74, human CYP3A4-like, in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP3A74 ablation was confirmed at the DNA, mRNA, and protein level. The subsequent characterization of the CYP3A74 KO clone highlighted significant transcriptomic changes (RNA-sequencing) associated with the regulation of cell cycle and proliferation, immune and inflammatory response, as well as metabolic processes. Overall, this study successfully developed a new CYP3A74 KO in vitro model by using CRISPR/Cas9 technology, which represents a novel resource for xenobiotic metabolism studies in cattle. Furthermore, the transcriptomic analysis suggests a key role of CYP3A74 in bovine hepatocyte cell cycle regulation and metabolic homeostasis.

Establishment and characterization of cytochrome P450 1A1 CRISPR/Cas9 Knockout Bovine Foetal Hepatocyte Cell Line (BFH12).

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.

Discovering the Protective Effects of Quercetin on Aflatoxin B1-Induced Toxicity in Bovine Foetal Hepatocyte-Derived Cells (BFH12).

Aflatoxin B1 (AFB1) induces lipid peroxidation and mortality in bovine foetal hepatocyte-derived cells (BFH12), with underlying transcriptional perturbations associated mainly with cancer, cellular damage, inflammation, bioactivation, and detoxification pathways. In this cell line, curcumin and resveratrol have proven to be effective in mitigating AFB1-induced toxicity. In this paper, we preliminarily assessed the potential anti-AFB1 activity of a natural polyphenol, quercetin (QUE), in BFH12 cells. To this end, we primarily measured QUE cytotoxicity using a WST-1 reagent. Then, we pre-treated the cells with QUE and exposed them to AFB1. The protective role of QUE was evaluated by measuring cytotoxicity, transcriptional changes (RNA-sequencing), lipid peroxidation (malondialdehyde production), and targeted post-transcriptional modifications (NQO1 and CYP3A enzymatic activity). The results demonstrated that QUE, like curcumin and resveratrol, reduced AFB1-induced cytotoxicity and lipid peroxidation and caused larger transcriptional variations than AFB1 alone. Most of the differentially expressed genes were involved in lipid homeostasis, inflammatory and immune processes, and carcinogenesis. As for enzymatic activities, QUE significantly reverted CYP3A variations induced by AFB1, but not those of NQO1. This study provides new knowledge about key molecular mechanisms involved in QUE-mediated protection against AFB1 toxicity and encourages in vivo studies to assess QUE's bioavailability and beneficial effects on aflatoxicosis.

Subcutaneous mast cell tumours: A prospective multi-institutional clinicopathological and prognostic study of 43 dogs.

BACKGROUND: Canine subcutaneous mast cell tumours (ScMCTs) reportedly have a good prognosis. However, biomarkers that can be used to predict outcome are currently limited. METHODS: A multicentre prospective study was conducted to identify new prognostic markers. Dogs with a first occurrence of ScMCT were enrolled upon primary tumour removal and regional lymphadenectomy. In the absence of metastasis, dogs were monitored, while dogs with overtly metastatic lymph nodes (histological node 3, HN3) received adjuvant vinblastine. RESULTS: Forty-three dogs were enrolled: 15 (34.9%) had at least one HN3 lymph node and received vinblastine, and 28 (65.1%) were monitored. Three tumours harboured exon 8 and 9 c-kit mutations. Eight (18.6%) dogs experienced tumour progression, and five (11.6%) died of MCT-related causes. The 1- and 2-year survival rates were 90% and 77%, respectively. Variables significantly associated with an increased risk of progression included high cytograde, a mitotic count (MC) greater than 4/10 high-power fields (hpf) and Ki67-index greater than 23. An MC greater than 4/10 hpf was also associated with an increased risk of tumour-related death. LIMITATIONS: Regional rather than sentinel lymphadenectomy was performed in these dogs. Dogs were enrolled in oncology referral centres, constituting a different population compared to previous studies. CONCLUSIONS: ScMCTs have a good prognosis. However, the metastatic rate at admission was higher in this study than previously reported, and a subset of tumours were associated with a fatal outcome despite multimodal treatment. Proliferative activity and cytograding may predict more aggressive behaviour in ScMCTs.

Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure?

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1.

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2.

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38ß MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38ß MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

Proposed Diagnostic Criteria and Classification of Canine Mast Cell Neoplasms: A Consensus Proposal.

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.

Discovering the Protective Effects of Resveratrol on Aflatoxin B1-Induced Toxicity: A Whole Transcriptomic Study in a Bovine Hepatocyte Cell Line.

Aflatoxin B1 (AFB1) is a natural feed and food contaminant classified as a group I carcinogen for humans. In the dairy industry, AFB1 and its derivative, AFM1, are of concern for the related economic losses and their possible presence in milk and dairy food products. Among its toxic effects, AFB1 can cause oxidative stress. Thus, dietary supplementation with natural antioxidants has been considered among the strategies to mitigate AFB1 presence and its toxicity. Here, the protective role of resveratrol (R) has been investigated in a foetal bovine hepatocyte cell line (BFH12) exposed to AFB1, by measuring cytotoxicity, transcriptional changes (RNA sequencing), and targeted post-transcriptional modifications (lipid peroxidation, NQO1 and CYP3A enzymatic activity). Resveratrol reversed the AFB1-dependent cytotoxicity. As for gene expression, when administered alone, R induced neglectable changes in BFH12 cells. Conversely, when comparing AFB1-exposed cells with those co-incubated with R+AFB1, greater transcriptional variations were observed (i.e., 840 DEGs). Functional analyses revealed that several significant genes were involved in lipid biosynthesis, response to external stimulus, drug metabolism, and inflammatory response. As for NQO1 and CYP3A activities and lipid peroxidation, R significantly reverted variations induced by AFB1, mostly corroborating and/or completing transcriptional data. Outcomes of the present study provide new knowledge about key molecular mechanisms involved in R antioxidant-mediated protection against AFB1 toxicity.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Curcumin Mitigates AFB1-Induced Hepatic Toxicity by Triggering Cattle Antioxidant and Anti-inflammatory Pathways: A Whole Transcriptomic In Vitro Study.

Aflatoxin B1 (AFB1) toxicity in livestock and human beings is a major economic and health concern. Natural polyphenolic substances with antioxidant properties have proven to be effective in ameliorating AFB1-induced toxicity. Here we assessed the potential anti-AFB1 activity of curcumin (pure curcumin, C, and curcumin from Curcuma longa, CL) in a bovine fetal hepatocyte-derived cell line (BFH12). First, we measured viability of cells exposed to AFB1 in presence or absence of curcumin treatment. Then, we explored all the transcriptional changes occurring in AFB1-exposed cells cotreated with curcumin. Results demonstrated that curcumin is effective in reducing AFB1-induced toxicity, decreasing cells mortality by approximately 30%. C and CL induced similar transcriptional changes in BFH12 exposed to AFB1, yet C treatment resulted in a larger number of significant genes compared to CL. The mitigating effects of curcuminoids towards AFB1 toxicity were mainly related to molecular pathways associated with antioxidant and anti-inflammatory response, cancer, and drug metabolism. Investigating mRNA changes induced by curcumin in cattle BFH12 cells exposed to AFB1 will help us to better characterize possible tools to reduce its consequences in this susceptible and economically important food-producing species.

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-Ο-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.

Insights into Aflatoxin B1 Toxicity in Cattle: An In Vitro Whole-Transcriptomic Approach.

Aflatoxins, and particularly aflatoxin B1 (AFB1), are toxic mycotoxins to humans and farm animal species, resulting in acute and chronic toxicities. At present, AFB1 is still considered a global concern with negative impacts on health, the economy, and social life. In farm animals, exposure to AFB1-contaminated feed may cause several untoward effects, liver damage being one of the most devastating ones. In the present study, we assessed in vitro the transcriptional changes caused by AFB1 in a bovine fetal hepatocyte-derived cell line (BFH12). To boost the cellular response to AFB1, cells were pre-treated with the co-planar PCB 3,3',4,4',5-pentachlorobiphenyl (PCB126), a known aryl hydrocarbon receptor agonist. Three experimental groups were considered: cells exposed to the vehicle only, to PCB126, and to PCB126 and AFB1. A total of nine RNA-seq libraries (three replicates/group) were constructed and sequenced. The differential expression analysis showed that PCB126 induced only small transcriptional changes. On the contrary, AFB1 deeply affected the cell transcriptome, the majority of significant genes being associated with cancer, cellular damage and apoptosis, inflammation, bioactivation, and detoxification pathways. Investigating mRNA perturbations induced by AFB1 in cattle BFH12 cells will help us to better understand AFB1 toxicodynamics in this susceptible and economically important food-producing species.

Nutrigenomic Effects of Long-Term Grape Pomace Supplementation in Dairy Cows.

The increasing demand for more animal products put pressure on improving livestock production efficiency and sustainability. In this context, advanced animal nutrition studies appear indispensable. Here, the effect of grape pomace (GP), the polyphenol-rich agricultural by-product, was evaluated on Holstein-Friesian cows' whole-blood transcriptome, milk production and composition. Two experimental groups were set up. The first one received a basal diet and served as a control, while the second one received a 7.5% GP-supplemented diet for a total of 60 days. Milk production and composition were not different between the group; however, the transcriptome analysis revealed a total of 40 genes significantly affected by GP supplementation. Among the most interesting down-regulated genes, we found the DnaJ heat-shock protein family member A1 (DNAJA1), the mitochondrial fission factor (MFF), and the impact RWD domain protein (IMPACT) genes. The gene set enrichment analysis evidenced the positive enrichment of 'interferon alpha (IFN-α) and IFN-γ response', 'IL6-JAK-STAT3 signaling' and 'complement' genes. Moreover, the functional analysis denoted positive enrichment of the 'response to protozoan' and 'negative regulation of viral genome replication' biological processes. Our data provide an overall view of the blood transcriptomic signature after a 60-day GP supplementation in dairy cows which mainly reflects a GP-induced immunomodulatory effect.

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

Longitudinal transcriptomic and genetic landscape of radiotherapy response in canine melanoma.

Canine malignant melanoma (MM) is a highly aggressive tumour with a low survival rate and represents an ideal spontaneous model for the human counterpart. Considerable progress has been recently obtained, but the therapeutic success for canine melanoma is still challenging. Little is known about the mechanisms beyond pathogenesis and melanoma development, and the molecular response to radiotherapy has never been explored before. A faster and deeper understanding of cancer mutational processes and developing mechanisms are now possible through next generation sequencing technologies. In this study, we matched whole exome and transcriptome sequencing in four dogs affected by MM at diagnosis and at disease progression to identify possible genetic mechanisms associated with therapy failure. According to previous studies, a genetic similarity between canine MM and its human counterpart was observed. Several somatic mutations were functionally related to MAPK, PI3K/AKT and p53 signalling pathways, but located in genes other than BRAF, RAS and KIT. At disease progression, several mutations were related to therapy effects. Natural killer cell-mediated cytotoxicity and several immune-system-related pathways resulted activated opening a new scenario on the microenvironment in this tumour. In conclusion, this study suggests a potential role of the immune system associated to radiotherapy in canine melanoma, but a larger sample size associated with functional studies are needed.

Comparative oncology: The paradigmatic example of canine and human mast cell neoplasms.

In humans, advanced mast cell (MC) neoplasms are rare malignancies with a poor prognosis. Only a few preclinical models are available, and current treatment options are limited. In dogs, MC neoplasms are the most frequent malignant skin tumours. Unlike low-grade MC neoplasms, high-grade MC disorders usually have a poor prognosis with short survival. In both species, neoplastic MCs display activating KIT mutations, which are considered to contribute to disease evolution. Therefore, tyrosine kinase inhibitors against KIT have been developed. Unfortunately, clinical responses are unpredictable and often transient, which remains a clinical challenge in both species. Therefore, current efforts focus on the development of new improved treatment strategies. The field of comparative oncology may assist in these efforts and accelerate human and canine research regarding diagnosis, prognostication, and novel therapies. In this article, we review the current status of comparative oncology approaches and perspectives in the field of MC neoplasms.

Validation of epigenetic mechanisms regulating gene expression in canine B-cell lymphoma: An in vitro and in vivo approach.

Despite canine B-cell Lymphoma (BCL) representing the most common haematological tumour, epigenetic events driving development and progression are scarcely known. Recently, canine Diffuse Large BCL (DLBCL) DNA methylome by genome-wide CpG microarray has identified genes and pathways associated to pathogenesis. To validate data previously obtained by array analysis, the CLBL-1 cell line was used and the HOXD10, FGFR2, ITIH5 and RASAL3 genes were selected. CLBL-1 cells were treated with two hypomethylating drugs (HDs; IC50, 50% inhibitory concentration), i.e. azacytidine and decitabine (DEC), either alone or in combination with three histone deacetylase inhibitors (HDACis; IC20), i.e. valproic acid, trichostatin and vorinostat. Following the incubation with both HDs, an overall decrease of promoter methylation was highlighted, thus confirming target genes hypermethylation. The highest mRNA restoration was observed following the exposure to HDs combined with HDACis, and mostly with valproic acid. Contrasting results were only obtained for RASAL3. An in vivo confirmation was finally attempted treating Nod-Scid mice engrafted with CLBL-1 cells with DEC. Although DEC did not arrest tumour growth, target genes promoter methylation was significantly reduced in DEC-treated mice vs controls. Overall, this work demonstrates that CLBL-1 cell line represents a reliable in vitro model to validate the methylation-dependent silencing of key genes for BCL; moreover, it may be useful for xenograft models in mice, despite its aggressive behaviour. In future, functional studies will be performed to deepen the role of selected genes on BCL pathogenesis and progression, and their methylation-dependent mechanism of regulation.