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

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.

Erratum: Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

[This corrects the article DOI: 10.1016/j.omtn.2023.04.004.].

Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

A gene-silencing platform (miQURE) has been developed and successfully used to deliver therapeutic microRNA (miRNA) to the brain, reducing levels of neurodegenerative disease-causing proteins/RNAs via RNA interference and improving the disease phenotype in animal models. This study evaluates the use of miQURE technology to deliver therapeutic miRNA for liver-specific indications. Angiopoietin-like 3 (ANGPTL3) was selected as the target mRNA because it is produced in the liver and because loss-of-function ANGPTL3 mutations and/or pharmacological inhibition of ANGPTL3 protein lowers lipid levels and reduces cardiovascular risk. Overall, 14 candidate miRNA constructs were tested in vitro, the most potent of which (miAngE) was further evaluated in mice. rAAV5-miAngE led to dose-dependent (≤-77%) decreases in Angptl3 mRNA in WT mice with ≤-90% reductions in plasma ANGPTL3 protein. In dyslipidemic APOE∗3-Leiden.CETP mice, AAV5-miAngE significantly reduced cholesterol and triglyceride levels vs. vehicle and scrambled (miSCR) controls when administrated alone, with greater reductions when co-administered with lipid-lowering therapy (atorvastatin). A significant decrease in total atherosclerotic lesion area (-58% vs. miSCR) was observed in AAV5-miAngE-treated dyslipidemic mice, which corresponded with the maintenance of a non-diseased plaque phenotype and reduced lesion severity. These results support the development of this technology for liver-directed indications.

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.

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.

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 17ß-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.

Significance of the goby Zosterisessor ophiocephalus as a sentinel species for Venice Lagoon contamination: Combining biomarker responses and bioaccumulation.

The Venice Lagoon is an interesting example of an ecosystem suffering for a considerable anthropogenic impact, resulting in high concentrations of persistent organic pollutants (POPs) in lagoon sediments and seafood. In this context, biomonitoring is a crucially important task. The present study aimed at evaluating the validity of a multiple biomarker approach in a benthic fish species. A total of 567 Zosterisessor ophiocephalus (Gobiidae) fish were collected in spring and autumn from three areas of Venice Lagoon (Porto Marghera, Val di Brenta, and Cà Roman) showing high, intermediate and low amounts of POPs, respectively. Aryl hydrocarbon receptor (AHR) and cytochrome P450 1A (CYP1A) mRNA levels, CYP1A protein amount and ethoxyresorufin O-deethylase activity (EROD) were measured in pooled liver and gills (mRNA levels only). Such biological data were then compared with polychlorinated biphenyls (PCBs) residues, measured in grass goby muscle by gas chromatography. Aryl hydrocarbon receptor and CYP1A mRNAs, protein and EROD were upregulated in accordance with PCB amounts measured in Z. ophiocephalus muscles. In fact, the highest AHR and CYP1A induction was observed in fish sampled in close proximity of the industrial area of Porto Marghera. Overall, the present study confirm the grass goby as a reliable sentinel species for Venice Lagoon, and AHR/CYP1A/EROD as a sensitive set of biomarkers of exposure for AHR ligands.

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.

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.

Effects of time culture and prototypical cytochrome P450 3A (CYP3A) inducers on CYP2B22, CYP2C, CYP3A and nuclear receptor (NR) mRNAs in long-term cryopreserved pig hepatocytes (CPHs).

In the present study, transcriptional and post-translational effects of culturing time and prototypical cytochrome P450 3A (CYP3A) inducers on principal nuclear receptors (NRs), CYP2B22, 2C and 3A were investigated in long-term stored (~10 years) cryopreserved pig hepatocytes (CPHs). In the time-course study, a crush and rise effect was observed for pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3) mRNAs, while a time-dependent increase of retinoid X receptor alpha (NR2B1) was noticed. Cytochrome P450 gene expression profiles were down-regulated as a function of time. In the induction study, an increase of NR1I2, NR1I3 and NR2B1 mRNAs was observed in dexamethasone-exposed CPHs. About CYPs, an overall up-regulation was seen in CPHs exposed to phenobarbital, while dexamethasone and rifampicin up-regulated only CYP3A. In both studies, transcriptional CYP results were confirmed at the post-translational level (immunoblotting and enzyme activities), except for CYP2B immunoblotting in the induction study. The present data demonstrate that long-term stored CPHs may be used to investigate mechanisms involved in CYPs regulation, expression and function; provide further info about NR regulation of CYPs, and confirm species-differences in these mechanisms of regulation; finally, they suggest the usefulness and relevance of gene expression profiling to early detect any modulation of CYP expression and bioactivity.

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 6ß-, 2ß- and 16ß-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.

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 17ß-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 3ß-hydroxysteroid dehydrogenase type 1 (HSD3ß1,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), HSD17ß3 (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.

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.