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

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

New molecular and therapeutic insights into canine diffuse large B cell lymphoma elucidates the role of the dog as a model for human disease

open21siopenAresu, Luca; Ferraresso, Serena; Marconato, Laura; Cascione, Luciano; Napoli, Sara; Gaudio, Eugenio; Kwee, Ivo; Tarantelli, Chiara; Testa, Andrea; Maniaci, Chiara; Ciulli, Alessio; Hillmann, Petra; Bohnacker, Thomas; Wymann, Matthias P; Comazzi, Stefano; Milan, Massimo; Riondato, Fulvio; Dalla Rovere, Giulia; Giantin, Mery; Giannuzzi, Diana; Bertoni, FrancescoAresu, Luca; Ferraresso, Serena; Marconato, Laura; Cascione, Luciano; Napoli, Sara; Gaudio, Eugenio; Kwee, Ivo; Tarantelli, Chiara; Testa, Andrea; Maniaci, Chiara; Ciulli, Alessio; Hillmann, Petra; Bohnacker, Thomas; Wymann, Matthias P; Comazzi, Stefano; Milan, Massimo; Riondato, Fulvio; Dalla Rovere, Giulia; Giantin, Mery; Giannuzzi, Diana; Bertoni, Francesc

Asiakaslähtöisen munasolunsaajien hoitoprosessin kehittäminen lapsettomuusklinikalla

Tämä opinnäytetyö oli työelämä lähtöinen kehittämistyö, joka toteutettiin Väestöliiton lapsettomuus klinikoiden Helsingin yksikössä. Tämän kehittämistyön tarkoituksena oli tuottaa uusi asiakaslähtöinen munansolunsaajien hoitoprosessi ja sen myötä parantaa asiakkaiden tyytyväisyyttä saamaansa hoitoon. Kehittämishankkeen tavoitteena oli kartoittaa haastattelemalla munasolunluovutushoidon läpi käyneiden asiakkaiden näkemyksiä nykyisestä hoitoprosessista ja sen asiakaslähtöisyydestä. Tavoitteena oli myös huomioida klinikan kokeneen henkilökunnan ajatuksia hoitoprosessin parantamiseksi. Uuden hoitoprosessin luominen aloitettiin määrittelemällä nykyinen munasolunsaajien hoitoprosessi. Tämän jälkeen kartoitettiin munasolunluovutushoidon läpi käyneiden asiakkaiden näkemyksiä nykyisestä hoitoprosessista, sen asiakaslähtöisyydestä ja heidän ehdotuksia hoitoprosessin parantamiseksi. Analysoitujen haastattelu aineistojen avulla tehtiin hahmotelma uudesta hoitoprosessista. Hahmotelma hoitoprosessista ja haastatteluiden tulokset esiteltiin klinikan henkilökunnalle, joka ideoi näiden pohjalta kehittämiskohteita uuteen hoitoprosessiin. Edellisten vaiheiden jälkeen kehitettiin lopullinen versio uudesta hoitoprosessista. Munasolunluovutushoito on otettu käyttöön Väestöliiton klinikoilla vuonna 1991 ja se on nykyisin yksi standardi lapsettomuudenhoito menetelmä. Munasolunluovutushoito on saajille iso panostus henkisesti ja taloudellisesti. Hoitoprosessin kehittämisellä pyritään siihen, että hoitoprosessin toimimattomuuden takia he eivät joutuisi uusien pettymysten eteen. Terveydenhuollossa on ymmärretty viime vuosina asiakaslähtöisyyden tärkeys niin Suomessa, kuin maailmallakin. Opinnäytetyön lähestymistapana oli toimintatutkimus. Aineistonkeruu menetelmänä oli munasolunluovutushoidossa olleiden asiakkaiden teemahaastattelu. Tämän lisäksi henkilökunnan ehdotuksia hoitoprosessin kehittämiseksi kartoitettiin aivoriihi ideoinnin avulla. Teemahaastatteluun osallistui kahdeksan asiakasta ja haastatteluaineisto analysoitiin induktiivisella sisällön analyysilla. Aivoriihi ideoinnin tuloksena syntyneet ideat pisteytettiin, ryhmiteltiin ja yhdisteltiin samaa tarkoittaviin ideoihin. Tämän jälkeen aineistosta muodostettiin uusi hoitoprosessi. Haastatteluaineiston tulosten mukaan asiakaslähtöisyys on empaattista vuorovaikutusta ja asiakkaan kokonaisuuden huomioimista. Asiakkaat kokivat nykyisen hoitoprosessin sujuvana, mutta toisaalta kokivat siihen sisältyvän hallitsemattomuutta. Hyvänä nykyisessä hoitoprosessissa he kokivat tulosten mukaan olevan empaattisen ja yksilöllisen kohtaamisen ja hoitoprosessin yksilöllisyyden. Monipuolisiin palveluihin, hoitoprosessin yksilölliseen suunnitteluun ja psyykkiseen taakkaan tulisi tulosten mukaan kiinnittää huomiota uutta hoitoprosessia laadittaessa. Suomessa ei ole tehty tutkimuksia asiakaslähtöisyydestä lapsettomuushoitoihin liittyen. Ulkomaisista tutkimuksista on saatu samansuuntaisia tuloksia asiakaslähtöisyydestä lapsettomuushoitoihin liittyen kuin tässä opinnäytetyössä. Se osoittaa samojen asioiden olevan tärkeitä lapsettomuuspotilaille maan rajoista huolimatta. Jatkossa Väestöliiton klinikoilla tulisi tehdä seurantaa, siitä miten uuden hoitoprosessin käyttöönotto on mennyt.This thesis is the result of a workplace based development project implemented at the Väestöliitto fertility clinic in Helsinki. The goal of the project was to develop a new patient centred ovum donation process which would increase patients’ satisfaction with their care. The aim of the project was to survey thoughts of patients who had gone through the ovum donation process with regard to the current treatment process and its patient centeredness. The expertise of the more experienced employees of the clinic was to be taken into consideration throughout the development project. The development of the new process began by defining the current care process for ovum donation recipients. Following this, the ovum donation recipients were interviewed for their thoughts of the current care process and its patient centeredness. The patients were asked for suggestions for the improvement of the care process. Following an analysis of the intelligence gathered from the interviews, an outline for a new patient care process was drafted. The analysis of the intelligence and the new patient care process were presented to the clinic employees at a staff event. The goal of the event was to encourage the employees to brainstorm ideas for improving patient centeredness and thus enabling them to take part in developing the improved patient care process. Following the event, with the new ideas gathered, the final care process was developed. The ovum donation treatments started in 1991 at Väestöliitto fertility clinic and it is today one of the standard treatment methods for infertility. The treatment method requires a significant financial and psychological input from the recipients. The patients treated by this method have usually suffered from infertility for several years already, and have experienced many disappointments whilst going through previous infertility treatments. The aim of developing the new, improved patient care process, is that a poor design of the care process would not add to the disappointments that the patient may experience. The value of patient centeredness has been realised in Finland and all over the world in the recent years. The approach for this thesis was an action research method. The data was collected by interviewing patients and by executing a brainstorming event with the personnel. Eight patients who had undergone ovum donation were interviewed by an individual theme interview method. The data was analysed by inductive content analysis. The new ideas collected during the brainstorming ses-sion were categorised into groups based on their similarities and differences. By using a points system, the ideas and categories were analysed and the most significant ideas were utilized in the design of the new care process. The results of the patient interviews show that patient centeredness is empathic interaction and entire consideration of the patient. The patients felt that the current care process was fluent, but uncontrollable. Positive things in the current treatment process were empathic and individual encounter with the caregivers and the individuality of the treatment process. According to analysed data, a diverse range of treatment methods, individual planning of the treatment process, and the psychological burden of the treatments are things which should be taken carefully into account when planning new treatment processes. Previous studies show similar results about patient centerednes

Effetti di fattori intrinseci ed estrinseci sull'espressione in vivo ed in vitro degli enzimi farmaco-metabolizzanti epatici del bovino

Cytochrome P450 superfamily (CYP) comprises an ubiquitous enzyme system, with the highest concentration found in the liver. These enzymes play a crucial role in the metabolism of xenobiotics and endogenous compounds. Their primary role is converting lipophilic compounds to more polar and hydrophilic metabolites by means of oxidative, reductive and hydrolitic reactions. Products of these chemical reactions can then be conjugated with polar endogenous compounds and readily excreted by the organism (Ioannides, 2006). The evaluation of liver biotrasformation pathways in veterinary species, but expecially in cattle, is considered very important, particularly for the presence of potentially harmful residues in foodstuff of animal origin (Sivapathasundaram et al., 2001). Thus, very few informations about bovine metabolism are actually available in literature and they primarily focused on catalytic activities and protein expression data (Sivapathasundaram et al., 2001; Nebbia et al., 2003; Szotáková et al., 2004; Dacasto et al., 2005). Recently, increasing importance has been given to toxicogenomics, the science which studies the correlations between genome structure, activity and toxicological effects of xenobiotics (Aardema and McGregor, 2002). The recent advances in molecular biology have resulted in the possibility to set up innovative, sensitive and specific biomolecular tecniques, that can be successfully applied in Drug Metabolism (DM) studies (Tugwood et al., 2003). As a consequence, the aim of the present research project consists on the application of some recent biomolecular techniques, as the quantitative Real Time PCR (Q RT-PCR), in order to study the expression and the regulation of some genes involved in bovine DM. The following methodological approach has been adopted: firstly a bibliographic search to identify genes involved in xenobiotics metabolism and then, collection of the corresponding available mRNA sequences from databases; clonage and sequencing of bovine sequences not yet available; primers design and set up of bovine-specific Q RT-PCR assays; total RNA isolation from cattle liver samples either of control or experimentally treated with illicit growth promoters; finally, the application of Q RT-PCR assays for the relative quantitation of selected transcripts (genes involved in xenobiotics metabolism and/or transcription factors) in: (a) beef cattle belonging to different meat cattle breeds; (b) veal calves and beef cattle illegally treated with dexamethasone (DEX) administered alone per os or intramuscularly, or in association with 17ß-oestradiol (E2: this last treatment has been considered only in beef cattle); (c) in primary cultures of bovine hepatocytes incubated with endogenous/exogenous precursors of steroids and in beef cattle illegally treated with the same molecules, in order to compare in vivo/in vitro effects. Messanger RNA results were successfully compared and/or correlated with catalytic activity data obtained by using marker substrates known as specific for each considered CYP isoform or phase II enzyme, but also with protein expression data (CYP1A, 2B, 2C, 2E and 3A immunoblotting and semiquantitative densitometric analysis). Partial sequences coding for bovine CYP2B, CYP4A, UGT1A4, RXR?, GR, HNF4? and 17ßHSDII genes (all involved in DM and its regulation) have been cloned, sequenced and submitted to GenBank; furthermore Q RT-PCR assays for a total of 28 transcripts have been designed and set up. Forementioned assays were applied to evaluate the hepatic biotransformative pattern in 18-20 months old beef cattle belonging to three different meat cattle breeds (Charolais, CH; Piedmontese, PM; Blonde d'Aquitaine, BA). Statistically significant differences (P<0.05 or less) have been noticed in CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C18, CYP3A4, UGT1A1, UGT1A6, UGT2B17, GSTA1, GSTM1, GSTP1 mRNA expression. In particular, CH presented the lowest mRNA expression for all the forementioned transcripts except for UGT1A6, if compared with the similar expression profile of PM and BA. Results obtained at the pre-transcriptional level were confirmed at the post-translational one by immunoblotting, only in the case of CYP2B and CYP3A: in this respect, protein expression data for these two enzymes demonstrated the same trend among breeds (CH<PM<BA). The in vitro metabolism of specific model substrates for each isoform, instead, did not agree with Q RT-PCR data (except in some instances), but, on the contrary, presented an opposite behaviour: in fact, CH appeared to be the breed gifted of the lower gene expression but of the higher biotransformation capability. This result could be probably ascribed to a more efficient catalytic system or to the presence of polymorphisms. Furthermore, the correlation analysis between single enzyme gene expression and the corresponding catalytic activity has been performed: for some isoforms a good correlation has been evidenced. Present results confirmed the breed as one of the internal factors that might modulate the metabolism in cattle. The same Q RT-PCR assays have been applied in other two experiences, where 15-18 months old beef cattle have been administered with illicit compounds; the aim of the present approach was to evaluate the effects of these substances on liver metabolism and to identify possible indirect biomarkers of treatment. In the first experiment, "Marchigiani" beef cattle were treated with DEX per os (TPD group) or intramuscularly (BPD group), whereas in the second study French crossbred cattle were administered DEX alone (Dde group) or in combination with E2 (DEde group). Gene expression data pointed out a significant induction of CYP1A1, CYP1A2 and CYP3A4 mRNA in DEde group, while a consistent inhibition of CYP2B6 e CYP2E1 was noticed in all treated groups. As regards the tested phase II enzymes, GSTA1 was induced in BPD and DEde groups, and SULT1A1 in TPD and DEde ones. Finally, among nuclear receptors, an increase either of CAR and RXR? expression in TPD and DEde groups or ER? (only in DEde group) was noticed. The design and the application of a 28 transcripts PCR-Array of hepatic metabolism in experiments regarding illicit treatments allowed the identification of some potential candidate genes that could be used in the future as indirect biomarkers on field, although only after a validation step. The same approach (only for some phase I enzymes and corresponding transcription factors) has been used to complete another experiment, in which veal calves were treated only with DEX per os or intramuscularly: the evidenced effects were completely different from other ones collected in beef cattle, probably for physiological differences (age, diet, anatomy and physiology of the gastro-enterical apparatus) that are likely to modulate DEX pharmacokinetics but also for difficulties strictly related with this particular kind of farming (stress conditions and iron-deprived diet). No statistically significant differences were observed in chosen CYPs and nuclear receptors gene expression, except for GR, induced in the group of animals treated intramuscularly with DEX. Furthermore, the CYP3A protein expression was inhibited in both treated groups, confirming the trend observed at both gene expression and catalytic activity levels. Finally, the effects of some endogenous/exogenous precursors of sexual steroids on some metabolism enzymes and transcription factors gene expression have been evaluated, by comparing one in vitro and one in vivo system. Primary cultures of bovine hepatocytes, incubated for 6 hours with 100 ’µM ADD or DHEA, and 15-18 months old beef cattle ("Valdostana" breed), treated with the same compounds at the dosage of 50 mg/pro capite once a week for 5 weeks, were used. Quantitative Real Time PCR results evidenced that the in vivo treatments with the forementioned molecules did not cause relevant effects (except for PXR and PPAR? inhibition in the group treated with DHEA), confirming both catalytic activities and immunoblotting results. On the contrary, in the in vitro system, DHEA caused a 1,5 - 4 fold induction of CYP2C9, GSTA1, DHEA-ST, 17ßHSD type II, CAR, PXR, RXR? and PPAR? mRNA expression, and a significant inhibition of CYP1A1, CYP2E1 and UGT2B17. The incubation with ADD, instead, evidenced only a significant inhibition of CYP1A1 expression. The different behavior observed in the in vivo and in vitro systems could be probably due to the dose applied and to the fact that the in vitro system represents a semplification of the organism in toto and that mimics with a lot of difficulties the pharmaco- and toxicological processes that normally happen in vivo (Freshney, 2001). Nonetheless, in vitro alternative methods are valid systems because they are rapid, riproducible and could substitute field experiments, that necessitate many animals. As a whole, other experiences, to standardize the protocol of hepatocytes isolation, are requested in order to reduce the inter-culture variability observed