Mauro Dacasto

Comparison of Hydrolytic and Conjugative Biotransformation Pathways in Horse, Cattle, Pig, Broiler Chick, Rabbit and Rat Liver Subcellullar Fractions

To complete a studyaimed at investigating the pattern of the basal activities of liver xenobioticmetabolizing enzymes in major and minor species intended for meat production, microsomal carboxylesterases and some conjugating enzyme activities were determined and compared in liver preparations from horses, cattle, pigs, rabbits and broiler chicks, using the rat as a reference species. Horses and broiler chicks exhibited a lower microsomal carboxylesterase activity towards indophenyl or p-nitrophenyl acetate than that measured in cattle or pig subfractions. Among food-producing species, the rate of glucuronidation of either 1-naphthol or p-nitrophenol was in the order pigs ∼rabbits > horses >> cattle > broiler chicks. The widest variations were observed in the acetylation capacity towards p-aminobenzoic acid or isoniazid, which in rabbits was 3-fold to 11-fold greater than that displayed by any other examined species; low but measurable activities were found in equine and bovine cytosols. The activity of cytosolic glutathione S-transferase (GST) accepting the general substrate 1-chloro-2,4-dinitrobenzene was significantly higher in rabbits, horses and pigs than in rat, broiler chicks and cattle. Finally, an uneven pattern of activity towards the other tested GST substrates – 3,4-dichloronitrobenzene, ethacrinic acid or 1,2-epoxybutane – was observed, possibly reflecting the species-related expression of different GST classes; in this respect, the conjugative capacity displayed by horses was higher than or comparable to that found in the other food-producing species.

Expression of Ki67, BCL-2, and COX-2 in Canine Cutaneous Mast Cell Tumors Association With Grading and Prognosis

The expression of Ki67, BCL-2, and COX-2 was investigated in 53 canine cutaneous mast cell tumors (MCTs) by immunohistochemistry and quantitative real time polymerase chain reaction (qPCR) to evaluate their prognostic significance and the association with the histologic grading and the mitotic index (MI). MCTs were graded according to the Patnaik grading system and the novel 2-tier grading system proposed by Kiupel. The numbers of mitotic figures/10 high-power fields (MI) were counted. Both grading systems were significantly associated with prognosis. The Patnaik grading was of limited prognostic value for grade 2 MCTs, with 23% being associated with mortality. The concordance among pathologists was strongly improved by the application of the 2-tier grading system, and 71% of high-grade MCTs were associated with a high mortality rate. MI and Ki67 protein expression were significantly associated with grading and survival. No significant association between BCL-2 protein expression and either grading system or health status was observed. BCL-2 mRNA expression was significantly higher in grade 2 than in grade 1 MCTs, while no statistically significant differences were detected between low- and high-grade MCTs. The increased BCL-2 mRNA level was significantly associated with increased mortality rate. The COX-2 protein expression was detected in 78% of the MCTs investigated. However, neither association with the tumor grade nor with the health status was observed. COX-2 mRNA was significantly up-regulated in MCTs compared to surgical margins and control skin tissue, but it was neither associated with tumor grade nor with survival.

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.

Effects of dexamethasone, administered for growth promoting purposes, upon the hepatic cytochrome P450 3A expression in the veal calf.

Dexamethasone (DEX) exerts its known anti-inflammatory and immunosuppressant activities through the interaction with the glucocorticoid receptor (GR). In human liver, DEX is metabolized by cytochrome P450 3A (CYP3A); moreover, it is among those xenobiotics which induce CYP3A itself. The transcriptional regulation of CYP3A involves GR and nuclear receptors (NRs). In cattle, DEX is used at low dosages as a growth promoter; besides, CYP3A is expressed in the liver. In the present study, the effects of two illicit DEX protocols upon liver CYP3A were investigated in the veal calf. Dexamethasone, administered per os (DOS) or injected intramuscularly (DIM) at growth promoting purposes, increased GR mRNA (+25.62% and +73.02% of CTRL for DOS and DIM, respectively), while tyrosine aminotransferase (TAT) and NRs gene expression profiles were unaffected; decreased CYP3A mRNA (-20.64% and -16.07% with Q RT-PCR; -30.55% and -34.31% with Northern blotting); at the post-translational level, decreased TAT activity (-19.84% and 44.34%), CYP3A apoprotein (-27.65% and -42.85%) and CYP3A-dependent enzyme activities (erythromycin N-demethylase, -78.89% and -23.87%; ethylmorphine N-demethylase, -44.26% and -28.37%; testosterone 6beta-hydroxylase, -44.60% and -18.07%; testosterone 2beta-hydroxylase, -43.95% and -11.69%); by contrast, an increase (about 2-fold) of the urinary 6beta-hydroxycortisol:cortisol ratio was observed in vivo. In summary, DEX modulates cattle liver CYP3A at pre- and post-translational level. Species-differences in GR-NRs-CYP3A regulation and in their response to differing DEX dosages might justify present results. Furthermore, the urinary 6beta-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals.

c-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumors: correlations with post-surgical prognosis

Cutaneous mast cell tumors (MCTs) are among the most common neoplasms in dogs and show a highly variable biologic behavior. Histological grading, cell proliferation markers, and KIT immunohistochemistry are typically used to predict post-surgical prognosis. In the present study, c-KIT messenger RNA (mRNA) expression was measured in canine MCTs and its relationship with tumor grade, immunohistochemical staining pattern, post-surgical prognosis, and mutations was investigated. A significant increase of c-KIT mRNA was observed in MCTs versus healthy skin and surgical margins. Mutations were observed in 8.3% of cases. The KIT staining pattern was investigated for both grading systems. In particular, staining pattern III was associated with grade II (G2) and G3 MCTs, while staining patterns I and II were associated with G1 and G2 MCTs. Considering the 2-tier histological grading, the high grade was mainly associated with pattern III (71%) while the low grade was associated with patterns II (70%) and I (28%). A weak association between the KIT staining pattern and outcome was also observed. The results obtained suggest that c-KIT mRNA is overexpressed in canine MCT, although the fold variations were not associated with the protein localization or complementary DNA mutations. These observations suggested that the 3 events were independent. The histological grading and the KIT staining pattern have prognostic value as previously published. Staining pattern I could be especially helpful in predicting a good prognosis of G2 MCTs. Sequence mutations were not necessarily suggestive of a worse prognosis, but might be useful in choosing a chemotherapy protocol.

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.

Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC

A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC.

Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6β-hydroxylation

Cultured rabbit hepatocytes were used to compare the relative activities of cytokines to inhibit the constitutive or rifampicin (RIF)-induced expression of the cytochrome P450 3A6 gene (CYP3A6). Human recombinant cytokines tested were interleukin-1beta (IL-1beta) (2 U/mL), interleukin-2 (IL-2) (5,000 U/mL) and interferon-gamma (IFN-gamma) (50 U/mL). Hepatocytes were cultured in the presence or absence of 25 microM RIF for 24 hr, with or without cytokines alone or in combination. All these cytokines inhibited RIF-induced P4503A6 expression without apparent cellular toxicity. By contrast, only IFN-gamma treatment provided a significant decrease (41%) in the constitutive P4503A6 protein level. Moreover, cytokines differed in their ability to repress RIF-dependent transcriptional induction of CYP3A6: IL-1beta and IL-2 were approximately equipotent, causing an almost 40-50% suppression of CYP3A6 mRNA and protein levels, whereas IFN-gamma exerted repressive effects only on P4503A6-related erythromycin N-demethylase activity and inducible protein expression. In fact, although strongly reducing P4503A6 protein content (an approximate 70% decrease), IFN-gamma did not exhibit any influence on CYP3A6 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1beta and IL-2 mainly promote a transcriptional repression mechanism, given the absence of effect of these cytokines on the basal P4503A6 level, whereas IFN-gamma exerts a post-transcriptional suppressive action on both induced and constitutive P4503A6 expression. Consequently, P4503A6-dependent progesterone 6beta-hydroxylase activity also presented a cytokine-specific pattern of inhibition, with a much greater sensitivity than P4503A6 immunoreactive protein to IL-1beta and IL-2 + IFN-gamma treatments. Thus, this study underlines the significant impact of inflammation on steroid metabolism.

Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics

Abstract The cytochrome P450 (CYP) superfamily constitutes a collection of enzymes responsible for the metabolism of a wide array of endo- and xenobiotic compounds. Much of the knowledge on substrate specificity and genetic identification of the various CYP isoforms is derived from research in rodents and humans and only limited information has been captured in the dog. Currently, there exist many gaps in our knowledge of canine CYP diversity as a result of the paucity of studies focusing on canine CYPs, canine CYP polymorphisms, and the therapeutic consequences of these genetic variants. Challenges engendered by this lack of information is further amplified by inter- and intraspecies differences in the specificity and affinity of substrates and inhibitors, prohibiting a simple extrapolation of probe substances used in human CYP research. This creates a need to develop and validate canine-specific CYP probes. Failure to understand this potential metabolic and pharmacogenomic diversity can also influence the interpretation of data generated in dogs to support human drug development. It is with these objectives in mind that we provide an overview of what is currently known about canine CYPs with the hope that it will encourage further exploration into this important area of research.