Vanessa Zancanella

Effects of illicit dexamethasone upon hepatic drug metabolizing enzymes and related transcription factors mRNAs and their potential use as biomarkers in cattle.

In cattle fattening, the illicit use of growth promoters (GPs) represents a major problem. The synthetic corticosteroid dexamethasone (DEX) is the GP mostly used, alone or in combination with other steroids or beta-agonists. Recently, GPs were shown to disrupt some cattle cytochromes P450 (CYPs) at the post-transcriptional level; therefore, the effects of two illicit protocols containing DEX (alone or together with 17beta-estradiol, 17betaE) upon main cattle liver drug metabolizing enzymes (DMEs) mRNAs and related transcription factors were investigated by quantitative real time RT-PCR. Eleven genes, out of the 18 considered, were significantly modulated by GPs. Corticosteroid-responsive genes did not respond univocally, whereas retinoic X receptor alpha (RXRalpha) and estrogen receptor alpha (ERalpha) were upregulated depending on the illicit protocol used. Nowadays, an increasing interest has been noticed toward the detection of biomarkers of response (BMRs) to be used in the screening of GPs misuse in cattle farming. In the present study, CYP2B6-like, CYP2E1, glutathione S-transferase A1- and sulfotransferase A1-like (GSTA1- and SULT1A1-like) mRNAs were significantly modulated regardless of the GP, the illicit protocol, and the animal breed, representing promising BMRs. The usefulness of these BMRs needs to be characterized more in depth.

Expression of Matrix Metalloproteinases, Tissue Inhibitors of Metalloproteinases and Vascular Endothelial Growth Factor in Canine Mast Cell Tumours.

Degradation of the extracellular matrix and angiogenesis are associated with tumour invasion and metastasis in human and canine neoplasia. Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and vascular endothelial growth factor-A (VEGF-A) are key mediators of these respective processes. Mast cell tumour (MCT) is the most common malignant cutaneous tumour in dogs. MCTs are always considered potentially malignant, but their true metastatic potential is unknown. In the present study, samples from seven grade 1, 22 grade 2 and six grade 3 MCTs were subjected to quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) to evaluate MMP-2, MMP-9, membrane-type 1 MMP (MT1-MMP), TIMP-2 and VEGF-A mRNA and protein expression. Gelatin zymography (GZ) was also performed to evaluate MMP-2 and MMP-9 activity. MMP-9 and VEGF-A mRNA increased with histological grade, while TIMP-2 decreased with increasing grade. Gene expression data obtained for MMP-9, VEGF-A and TIMP-2 were confirmed by IHC for evaluation of the respective proteins. In contrast, MMP-2 and MT1-MMP had variable, but similar, expression for both mRNA and protein. Despite the high variability observed, there was correlation between MMP-2 and MT1-MMP mRNA expression (r=+0.91, P<0.0001). The MMP-2:TIMP-2 and MMP-9:TIMP-1 mRNA ratios showed an imbalance between MMPs and their specific inhibitors in MCTs, which increased with the histological grade. Finally, the activities of both latent and active forms of MMP-2 and MMP-9 were evaluated by GZ and there were significant increases in their activities with increasing histological grade and immunohistochemical expression. This study demonstrates that MMP-9, TIMP-2 and VEGF-A expression is related to histological grade and suggests that these markers are possible indicators of malignancy and targets for therapeutic strategies.

Matrix metalloproteinases and their inhibitors in canine mammary tumors

BackgroundMalignant 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.ResultsMMP-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.ConclusionsOur 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.

Steroidogenic enzyme gene expression profiles in the testis of cattle treated with illicit growth promoters

Recently, the effect of illicit growth promoters (GPs) upon the cattle transcriptome has drawn the increasing attention of the scientific community. In the present study, the pre-transcriptional effects of three different illicit protocols on a set of target genes, including steroidogenic enzymes and three related transcription factors, were estimated in cattle testis. Beef cattle were administered with dexamethasone (DEX) orally (group D(1)) or intramuscularly in experiment 1 (group DIM). In experiment 2, DEX was orally administered alone (group D(2)) or with 17β-estradiol (group DE), and in experiment 3, dehydroepiandrosterone and boldione were orally administered alone (group DHEA and group ADD) or in combination (group DHAD). The GP effects were measured by quantitative real time RT-PCR. The results of our study were significant but not univocal. A GP-dependent effect on target gene mRNA levels was noticed for 3β-hydroxysteroid dehydrogenase type 1 (HSD3β1,p<0.05 and p<0.01 for the D(2), DE and DHAD groups, respectively), the cytochrome P450 side chain cleavage (DHAD, p<0.05), the cytochrome P450 17A1 (DIM and D(2), p<0.05), HSD17β3 (DE, p<0.05), aromatase (DHEA, p<0.05), the androgen receptor (DHAD, p<0.05) and the mineralocorticoid receptor-like (DIM, p<0.05). Our present results suggest that different GP schedules are likely to affect genes involved in steroid synthesis and regulation in cattle testis. Thus, this tissue might be considered a potential surrogate tissue that warrants further study into its usefulness in the screening of GP abuse.

Constitutive expression of drug metabolizing enzymes and related transcription factors in cattle testis and their modulation by illicit steroids

In veterinary species, little information about extrahepatic drug metabolism is actually available. Therefore, the presence of foremost drug metabolizing enzymes (DMEs) and related transcription factors mRNAs was initially investigated in cattle testis; then, their possible modulation following the in vivo exposure to illicit growth promoters (GPs), which represent a major issue in cattle farming, was explored. All target genes were expressed in cattle testis, albeit to a lower extent compared to liver ones; furthermore, illicit protocols containing dexamethasone and 17β-oestradiol significantly up-regulated cytochrome P450 1A1, 2E1, oestrogen receptor-α and peroxisome proliferator-activated receptor-α mRNA levels. Overall, the constitutive expression of foremost DMEs and related transcription factors was demonstrated for the first time in cattle testis and illicit GPs were shown to affect pre-transcriptionally some of them, with possible consequences upon testicular xenobiotic drug metabolism.

Proposed new nomenclature for Bos taurus cytochromes P450 involved in xenobiotic drug metabolism

The cytochrome P450 (CYP) superfamily of drug metabolizing enzymes (DMEs) plays a central role in the oxidative metabolism of xenobiotics to which living organisms are exposed. In Bos taurus (cattle), a definitive nomenclature for CYP proteins is still lacking, and to unambiguously settle cattle nomenclature a phylogenetic analysis of proteins belonging to CYP 1-4 families was performed. Sequences collected from GenBank and Dr Nelsons P450 homepage databases were analyzed according to the maximum likelihood method. Phylogenetic outputs showed that CYPs sharing the same name and collected from different species did not form, in several instances, monophyletic groups. Some cattle CYPs did not group with their supposed human orthologous counterparts, thus requiring a new nomenclature. Name changes mostly mirrored the orthologous counterparts established for other species, and new names were created when no clear orthologous sequences were identified. The new nomenclature will allow a more appropriate investigation of biochemical and molecular mechanisms involved in the expression and regulation of these DMEs.

Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.

Cattle hepatocytes have already been used in veterinary in vitro toxicology, but their usefulness as a multi-parametric screening bioassay has never been investigated so far. In this study, cattle hepatocytes were incubated with illicit steroids/prohormones (boldenone, BOLD; its precursor boldione, ADD; dehydroepiandrosterone, DHEA; an association of ADD:BOLD), to characterize their transcriptional effects on drug metabolizing enzymes (DMEs) and related nuclear receptors (NRs), on cytochrome P450 3A (CYP3A) apoprotein and catalytic activity as well as to determine ADD and BOLD metabolite profiling. DHEA-exposed cells showed an up-regulation (higher than 2.5-fold changes) of three out of six NRs, CYP2B22 and CYP2C87; likewise, ADD:BOLD increased CYP4A11 mRNA levels. In contrast, a reduction of CYP1A1 and CYP2E1 mRNAs (lower than 2.5(-1)-fold changes) was noticed in ADD- and DHEA-incubated cells. No effect was noticed on CYP3A gene and protein expression, though an inhibition of 6β-, 2β- and 16β-hydroxylation of testosterone (higher than 60% of control cells) was observed in ADD- and BOLD-exposed cells. Finally, 17α-BOLD was the main metabolite extracted from hepatocyte media incubated with ADD and BOLD, but several mono-hydroxylated BOLD and ADD derivatives were detected, too. Collectively, cattle hepatocytes can represent a complementary screening bioassay, useful to characterize growth promoters metabolite profiling and their effects upon DMEs expression, regulation and function.

Constitutive expression and phenobarbital modulation of drug metabolizing enzymes and related nuclear receptors in cattle liver and extra-hepatic tissues

In humans and rodents, phenobarbital (PB) induces hepatic and extra-hepatic drug metabolizing enzymes (DMEs) through the activation of specific nuclear receptors (NRs). In contrast, few data about PB transcriptional effects in veterinary species are available. The constitutive expression and modulation of PB-responsive NR and DME genes, following an oral PB challenge, were investigated in cattle liver and extra-hepatic tissues (duodenum, kidney, lung, testis, adrenal and muscle). Likewise to humans and rodents, target genes were expressed to a lower extent compared to the liver with few exceptions. Phenobarbital significantly affected hepatic CYP2B22, 2C31, 2C87, 3A and UDP-glucuronosyltransferase 1A1-like, glutathione S-transferase A1-like and sulfotransferase 1A1-like (SULT1A1-like) mRNAs and apoprotein amounts; in extra-hepatic tissues, only duodenum showed a significant down-regulation of SULT1A1-like gene and apoprotein. Nuclear receptor mRNAs were never affected by PB. Presented data are the first evidence about the constitutive expression of foremost DME and NR genes in cattle extra-hepatic tissues, and the data obtained following a PB challenge are suggestive of species-differences in drug metabolism; altogether, these information are of value for the extrapolation of pharmacotoxicological data among species, the characterization of drug–drug interactions as well as the animal and consumer’s risk caused by harmful residues formation.

Feline intestinal mast cell tumours: clinicopathological characterisation and KIT mutation analysis.

Objectives Feline intestinal mast cell tumours (FIMCTs) are rare and reportedly characterised by poor differentiation, aggressive biological behaviour and lack of reliable therapeutic aids. KIT proto-oncogene-activating mutations have never been investigated in these tumours. This study describes the main clinicopathological and microscopic features observed in 17 FIMCTs. Methods Tumour degree of differentiation, proliferative activity, Kit protein expression and KIT mutations were evaluated and correlated with survival to assess their prognostic relevance. Results Ten tumours were located in the small intestine, two in the ileocaecocolic junction, and five in the large intestine. Survival times ranged from 3–538 days. Fifteen tumours were evaluated histologically, and there were six well-differentiated, six moderately differentiated and three poorly differentiated FIMCTs. The last showed a medium-to-large deposition of collagen tissue (P <0.001), and significantly higher mitotic and Ki67 indexes compared with more differentiated tumours (P = 0.011). On survival analysis, tumour degree of differentiation (P <0.001) and a mitotic index >2 (P = 0.022) were significantly associated with decreased survival times. Twelve cases showed Kit protein immunoexpression. The Kit pattern was membranous in five cases (33.3%), focal paranuclear in five (33.3%) and diffuse cytoplasmic in two (13.3%). Cytoplasmic Kit patterns were associated with a lesser differentiation (P = 0.015). Mutation analysis was successfully performed on 12 primary tumours and four lymph node metastases; however, no encoding mutation was detected. Conclusions and relevance Contrary to reports in the literature, FIMCTs seem to have an extremely variable biological behaviour. We propose a classification based on tumour degree of differentiation and proliferative activity. These findings need to be confirmed in larger series, and exploration of further genomic regions of KIT is warranted to clarify its role in the development and progression of these neoplasms.

Characterization of ligand-dependent activation of bovine and pig constitutive androstane (CAR) and pregnane X receptors (PXR) with interspecies comparisons.

Abstract 1. Nuclear receptors CAR (NR1I3) and PXR (NR1I2) are major ligand-activated transcriptional regulators of xenobiotic metabolism and disposition and modulators of endobiotic metabolism. Differences in xenobiotic selectivity between the human and rodent receptors are well recognized but there is lack of such information on properties of CAR and PXR in important domestic animals. 2. The pig and bovine receptors were cloned and their ligand profiles were systematically compared to corresponding human and mouse forms utilizing a panel of xenobiotics and structural analysis. 3. Pig CAR and PXR resemble their human counterparts which can be rationalized by only modest amino acid changes between critical residues of the human ligand-binding pockets (H203Q for CAR, L210V and M243I for PXR). 4. In contrast, bovine CAR shows a blunted response to CAR agonists and inverse agonists. These changes are likely due to disruptive mutations at or near critical hydrogen bond-forming residues (N165I, Y326F). The unresponsiveness of bovine PXR to human- and mouse-selective agonists may be related to substitutions at important ligand-contacting residues R410Q and F305V, respectively. 5. Our findings have implications for regulation of drug-metabolizing enzymes and transporters and pharmacokinetics in cattle and pigs.