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.

Development and optimization of a fentanyl pharmacokinetic model for target-controlled infusion in anaesthetized dogs.

OBJECTIVE: To investigate pharmacokinetics (PK) of fentanyl administered by target-controlled infusion (TCI), and to develop a PK model optimized by covariates for TCI in anaesthetized dogs. STUDY DESIGN: Prospective clinical study. ANIMALS: A group of 20 client-owned dogs with spinal pain undergoing anaesthesia for magnetic resonance imaging. METHODS: Fentanyl was administered as an infusion to 20 anaesthetized dogs using a TCI system incorporating a previously described fentanyl two-compartment PK. Arterial blood samples were collected at specific time points during the infusion and over 60 minutes post-infusion for measurement of fentanyl plasma concentrations. The predictive performance of the Sano PK model was assessed by comparing predicted and measured plasma concentrations. A population PK analysis was then performed using a nonlinear mixed-effect modelling approach, allowing inter- and intra-individual variability estimation. Finally, a quantitative stepwise evaluation of the influence of various covariates such as weight, body condition score, size, size-related age, sex and type of premedication on the PK model was considered. RESULTS: Overall predictive performance of the Sano PK set of variables was not clinically acceptable in anaesthetized dogs. Fentanyl PK was best described by a three-compartment model. Weight and sex were found to affect the volume of distribution of the central compartment. Addition of these two covariate/variable associations resulted in a reduction of the objective function value (OFV) from -340.18 to -448.34, and of the median population weighted residual and the median population absolute weighted residual from 16.1% and 38.6% to 3.9% and 20.3%, respectively. Fentanyl infusions at measured concentrations up to 5.4 ng mL-1 in sevoflurane-anaesthetized dogs resulted in stable anaesthesia and smooth recoveries without complications. CONCLUSIONS AND CLINICAL RELEVANCE: A population three-compartment PK model for fentanyl TCI in anaesthetized dogs was developed. Weight and sex have been detected and incorporated as significant covariates.

Natural contaminants in bee pollen: DNA metabarcoding as a tool to identify floral sources of pyrrolizidine alkaloids and fungal diversity.

The use of bee pollen as a food supplement has increased in recent years as it contains several nutrients and phytochemicals. However, depending on floral composition, bee pollen can be contaminated by pyrrolizidine alkaloids (PAs), PA N-oxides (PANOs) and toxigenic fungi found in plants, which may pose a potential health risk for consumers. Thus, a DNA metabarcoding approach based on internal transcribed spacer 2 (ITS2) region was used to identify the plant sources of 17 PAs/PANOs detected by a validated method in liquid chromatography coupled to mass spectrometry (LC-MS/MS), as well as floral and fungal diversity in 61 bee pollen samples. According to LC-MS/MS analysis, 67% of the samples contained PAs/PANOs with mean concentration of 339 µg/kg. The contamination pattern was characterised by lycopsamine- and senecionine-type PAs/PANOs. PA/PANO-producing plants were identified in 54% of the PA/PANO-contaminated samples analysed by DNA metabarcoding, which also allowed identifying the overall floral and fungal composition of 56 samples. To evaluate the performance of the molecular approach, a subset of 25 samples was analysed by classical palynology. Palynological analysis partially confirmed the results of DNA metabarcoding, which had a better performance in distinguishing pollens of different genera from Asteraceae (76%) and Brassicaceae (88%). However, the molecular analysis did not identify pollens from Castanea, Eucalyptus, Hedera and Salix, which were abundant in 11 samples according to palynology. On the other hand, the molecular analysis allowed identifying several fungal genera in 33 samples, including the toxigenic fungi Alternaria and Aspergillus, which were positively correlated to the plant genus Hypericum. Despite limitations in identifying some pollen types, these preliminary results suggest that the DNA metabarcoding could be applied in a multidisciplinary approach to give a picture of floral and fungal diversity, which can be sources of natural contaminants in bee pollen and would help to control its safety.

Dexmedetomidine and ketamine simultaneous administration in tigers (Panthera tigris): pharmacokinetics and clinical effects.

BACKGROUND: The study determines the pharmacokinetic profiles of dexmedetomidine (DEX), ketamine (KET) and its active metabolite, norketamine (NORKET), after simultaneous administration. Moreover, the study evaluates the sedative effects of this protocol, its influence on the main physiological variables and the occurrence of adverse effects. METHODS: Eighteen captive tigers were initially administered with a mixture of DEX (10 µg/kg) and KET (2 mg/kg) by remote intramuscular injection. In case of individual and specific needs, the protocol was modified and tigers could receive general anaesthesia, propofol or additional doses of DEX and KET. RESULTS: Based on the immobilisation protocol, nine animals were assigned to the standard protocol group and the other nine to the non-standard protocol group. Higher area under the first moment curve (AUMC0-last) and longer mean residence time (MRT0-last) (P<0.05) were observed in the non-standard protocol group for DEX, KET and NORKET, and higher area under the concentration-time curve from administration to the last measurable concentration (AUC0-last) only for KET. The KET metabolisation rate was similar (P=0.296) between groups. No differences between groups were detected in terms of stages of sedation and recoveries. All physiological variables remained within normality ranges during the whole observation period. During the hospitalisation period, no severe adverse reactions and signs of resedation were observed. CONCLUSION: The simultaneous administration of 10 µg/kg of DEX and 2 mg/kg of KET can be considered an effective protocol for chemical immobilisation of captive tigers, along with dosage adjusments or when other drugs are needed.

Microbial metabarcoding highlights different bacterial and fungal populations in honey samples from local beekeepers and market in north-eastern Italy.

Due to its chemical properties, honey does not foster the growth of microorganisms, however it may contain a rich microbial community, including viable, stressed, and not viable microbes. In order to characterize honey microbiota focusing on the difference between products from beekeepers and large retail in the present study a culture-independent approach based on DNA metabarcoding was applied. Honey samples were collected from Local Beekeepers (LB) and Market sales (M) during four years with the aim to investigate the microbiological quality in the honey market. Extraction and amplification of DNA from honey samples showed reduced efficiency with increasing age of honey, with the loss of 50-80% of samples four years old (2014). For this reason, only samples of similar age were compared and the analysis of microbial communities focused on year 2017, for a total of 75 samples. Differences in alpha and beta-diversity were evidenced comparing microbial communities between LB and M samples. In particular, contaminant bacteria dominated the microbiota in M samples while LB samples were enriched in Lactic Acid Bacteria (LAB) that cannot be isolated with culture-dependent approaches.

Pyrrolizidine alkaloids in bee pollen identified by LC-MS/MS analysis and colour parameters using multivariate class modeling.

Toxic pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) can be present in bee pollen depending on the plants visited by bees. A liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated to monitor 17 PAs/PANOs in 44 bee pollens. The CIE-L∗a∗b∗ colour coordinates with the specular component either included or excluded were recorded in pellets and ground aliquots. Lightness (L∗) and yellowness (b∗) of ground bee pollen were significantly correlated to PAs/PANOs content. The L∗ and b∗ cut-offs sorted by a receiver operating characteristic analysis to predict PAs/PANOs presence showed a significant increase in the relative risk to detect amounts higher than 84 µg kg-1. Two supervised canonical discriminant analyses confirmed that pollen without PAs could be distinguished from those containing PAs/PANOs. The data suggest that instrumental colour coupled with supervised models could be used as a screening test for PAs/PANOs in bee pollen, before the confirmatory LC-MS/MS analysis.

Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle.

Cytochrome P450 3A is the most important CYP subfamily in humans, and CYP3A4/CYP3A5 genetic variants contribute to inter-individual variability in drug metabolism. However, no information is available for bovine CYP3A (bCYP3A). Here we described bCYP3A missense single nucleotide variants (SNVs) and evaluated their functional effects. CYP3A28, CYP3A38 and CYP3A48 missense SNVs were identified in 300 bulls of Piedmontese breed through targeted sequencing. Wild-type and mutant bCYP3A cDNAs were cloned and expressed in V79 cells. CYP3A-dependent oxidative metabolism of testosterone (TST) and nifedipine (NIF) was assessed by LC-MS/MS. Finally, SNVs functional impact on TST hydroxylation was measured ex vivo in liver microsomes from individually genotyped animals. Thirteen missense SNVs were identified and validated. Five variants showed differences in CYP3A catalytic activity: three CYP3A28 SNVs reduced TST 6ß-hydroxylation; one CYP3A38 variant increased TST 16ß-hydroxylation, while a CYP3A48 SNV showed enhanced NIF oxidation. Individuals homozygous for rs384467435 SNV showed a reduced TST 6ß-hydroxylation. Molecular modelling showed that most of SNVs were distal to CYP3A active site, suggesting indirect effects on the catalytic activity. Collectively, these findings demonstrate the importance of pharmacogenetics studies in veterinary species and suggest bCYP3A genotype variation might affect the fate of xenobiotics in food-producing species such as cattle.

Clinical pharmacokinetics of a dexmedetomidine-methadone combination in dogs undergoing routine anaesthesia after buccal or intramuscular administration.

This study aimed to define the pharmacokinetic profiles of dexmedetomidine and methadone administered simultaneously in dogs by either an oral transmucosal route or intramuscular route and to determine the bioavailability of the oral transmucosal administration relative to the intramuscular one of both drugs, so as the applicability of this administration route in dogs. Twelve client-owned dogs, scheduled for diagnostic procedures, were treated with a combination of dexmedetomidine hydrochloride (10 µg/kg) and methadone hydrochloride (0.4 mg/kg) through an oral transmucosal route or intramuscularly. Oral transmucosal administration caused ptyalism in most subjects, and intramuscular administration caused transient peripheral vasoconstriction. The results showed reduced and delayed absorption of both dexmedetomidine and methadone when administered through an oral transmucosal route, with median (range) Cmax values of 0.82 (0.42-1.49) ng/ml and 13.22 (2.80-52.30) ng/ml, respectively. The relative bioavailability was low: 16.34% (dexmedetomidine) and 15.5% (methadone). Intramuscular administration resulted in a more efficient absorption profile, with AUC and Cmax values for both drugs approximately 10 times higher. Dexmedetomidine and methadone administered simultaneously by an oral transmucosal route using injectable formulations were not well absorbed through the oral mucosa. Nevertheless, additional studies on these drugs combination using alternative administration routes are recommended.

Assessment of antibacterial drug residues in milk for consumption in Kosovo.

The objective of this study was to assess the occurrence of drug residues in the raw milk collected from individual farms and milk collection points during 2009-2010 in six different major regions of Kosovo (Prishtinë, Gjilan, Mitrovicë, Pejë, Gjakovë, Prizren). In the present study, a total of 1734 raw milk samples were collected, and qualitatively screened with two different tests, the Delvotest SP assay and an enzyme-linked receptor-binding assay (SNAP). Overall, 106 (6.11%) out of 1734 samples examined with Delvotest SP contained possible drug residues (5.12% and 7.51% of samples from 2009 and 2010, respectively). All suspect samples were further analyzed by three distinct enzyme-linked receptor-binding assays specific for ß-lactams (new ß-lactam test), tetracyclines (SNAP tetracycline test), and sulfonamides (SNAP sulfamethazine test). Only the new SNAP ß-lactam test detected residues in 40 out of 52 samples in 2009 and 54 out of 54 suspect samples in 2010. A confirmatory method based on liquid chromatography-tandem mass spectrometry was used to confirm the presence of ß-lactam drug residues in samples detected by the enzyme-linked receptor-binding assay. Amoxicillin, penicillin G, and cloxacillin were the most frequently detected residues and were in a concentration range between 2.1 µg/kg and 1973 µg/kg. Seventeen of the positive samples exceeded the maximum residue levels for one or more ß-lactam drug. The highest number of positive milk samples came from the Pejë Region (58.8%) and Gjakovë Region (23.5%), and the lowest number of positive samples originated from Gjilan (5.88%), with no positive samples detected in two regions, Mitrovicë and Prizren.

Approved medication of water with enrofloxacin to treat turkey colibacillosis: assessment of efficacy using a PK/PD approach.

The efficacy of administering enrofloxacin at 10mg/kg in medicated water to turkeys was evaluated by applying a PK/PD approach to the kinetic parameters obtained after oral pulsed administration and to the MIC values of avian pathogenic Escherichia coli (APEC) strains isolated from commercial turkey flocks. The kinetic parameters of enrofloxacin were evaluated in 10 healthy and 10 colisepticemic turkeys that received the drug dissolved in medicated water at 89 µg/mL and 71 µg/mL, respectively, for 10h/day for 5 days. Blood samples were collected for 24h from all turkeys on the last day of treatment, and the serum was analysed by HPLC with fluorimetric detection. The mean AUC (7374.53±1067.64 h ng/mL and 7656.95±1460.61 h ng/mL) and C(max) values (673.09±186.18 ng/mL and 543.50±68.75 ng/mL) obtained for healthy and sick turkeys were not significantly different. High-level resistance was observed in 30.3% of strains, 40.5% exhibited intermediate resistance, and only 29.2% were susceptible; the MIC(50) and MIC(90) values were 1mg/L and 32 mg/L, respectively. The PK/PD parameters C(max)/MIC(50) (0.67 and 0.54 for healthy and sick turkeys, respectively) and AUC/MIC(50) (7.37 and 7.66) were lower than the efficacy breakpoints reported for fluoroquinolones. These results indicate that authorised dosage of enrofloxacin used in pulsed medicated water administration could be ineffective against more than the 70% of circulating APEC strains, indicating the need to test the drug susceptibility of APEC prior to administering the drug and adopting a more convenient medication schedule for mass treatment.

Metal ion-mediated assembly of effective phenanthroline-based G-quadruplex ligands.

Transition metal ions can drive the assembly of small planar ligands to produce structures able to efficiently recognize G-quadruplex DNA arrangements.

Perylene side chains modulate G-quadruplex conformation in biologically relevant DNA sequences.

The stabilisation of different G-quadruplex intra- and intermolecular structures by a number of perylene derivatives characterised by side chains ending with linear or cyclic amines was investigated by electrophoretic (EMSA) and spectroscopic (CD) techniques. The G-rich sequences included the biologically relevant human telomeric TTAGGG runs and the NHE region of the c-myc oncogene. The test compounds could be subdivided into two families: derivatives carrying a cyclic amine in the side chains, which show a reduced binding to the G-quadruplex form, and linear amine congeners, exhibiting enhanced affinity. The latter efficiently induce pairing of multiple DNA chains, while the former are not able to overcome the original folding of the nucleic acid sequence which is preserved in the complex. Remarkably, addition of the perylenes to G-rich sequences paired in a double helical form results in G-quadruplex induction by weak binders only. This is likely related to the ability of strong G-quadruplex binders, but not of weak G-quadruplex binders, to efficiently intercalate into the double-stranded arrangement, which becomes stabilised and is not prone to undergo denaturation and subsequent G-quadruplex folding essentially for kinetic reasons. Hence, two apparently conflicting requirements emerge from this work. In fact, linear alkylamino terminals in the perylene side chains are capable of strong and selective G-quadruplex recognition, but only cyclic amine end groups favour duplex-quadruplex transitions that are likely crucial to produce biological and pharmacological effects in living systems.

Aminoacyl-anthraquinone conjugates as telomerase inhibitors: synthesis, biophysical and biological evaluation.

The telomerase-telomere complex is a prospective anticancer target. To inhibit enzyme activity by induction of G-quadruplex in human telomeres, we have synthesized a small library of 2,6- and 2,7-amino-acyl/ peptidyl anthraquinones with diverse connecting linkers, charge, lipophilicity and bulk. The test compounds modulated G-quadruplex stability to different extents and showed clear preference for quadruplex over duplex DNA. Telomerase inhibition correlated with G-quadruplex stabilization. A SAR analysis showed that type of linkage between the linker and the anthraquinone, together with the position of the side chains and the nature of the amino acid components play a major role both in stabilizing G-quadruplex and producing telomerase inhibition. Short-term cytotoxic activity was poor. However, after prolonged exposure to effective G-quadruplex binders, cells became senescent. These results are of help in the rational design of more efficient G-quadruplex stabilizers, possibly endowed with cancer cell-selective antiproliferative effects.

Effective DNA inhibitors of cathepsin g by in vitro selection.

Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions.

Tri-, tetra- and heptacyclic perylene analogues as new potential antineoplastic agents based on DNA telomerase inhibition.

A recent approach in anticancer chemotherapy envisages telomerase as a potentially useful target. An attractive strategy deals with the development of compounds able to stabilize telomeric DNA in the G-quadruplex folded structure and, among them, a prominent position is found in the perylenes. With the aim to further investigate the role of drug structure, in view of possible pharmaceutical applications, we synthesized a series of compounds related to PIPER, a well-known perylene-based telomerase inhibitor. We modified the number of condensed aromatic rings and introduced different side chains to modulate drug protonation state and extent of self-aggregation. Effective telomerase inhibition was induced by heptacyclic analogues only, some showing a remarkably wide selectivity index with reference to inhibition of Taq polymerase. G-quadruplex stabilization was monitored by circular dichroism and melting experiments. Cell cytotoxicity measurements indicated a poor short-term cell killing ability for the best G-quartet binders. Besides the presence of a planar seven-condensed ring system, the introduction of a cyclic amine in the side chains critically affects the selectivity window.

Interactions of low-molecular-weight semi-synthetic sulfated heparins with human leukocyte elastase and human Cathepsin G.

Semi-synthetic low-molecular-weight heparin samples (LMWHs), having homogeneous degree of polymerization and saccharide backbone, but differing in the number and location of sulfate groups, were investigated in their ability to interfere with the pharmacologically relevant targets human leukocyte elastase (EL) and human Cathepsin G (CatG). Spectroscopic studies were performed for a quantitative evaluation of the enzyme-inhibitor dissociation constant, K(i), and of the IC(50) values for the inhibition of cleavage of target peptide sequences. Both proteases are inhibited by the tested polysaccharides through a mixed hyperbolic binding process. A non-linear relationship was found between degree of sulfation and binding affinity or enzyme inhibition properties, showing a composite correlation between heparin charge density and interference with EL/CatG activity.

Toward efficient Zn(II)-based artificial nucleases.

A series of cis-cis-triaminocyclohexane Zn(II) complex-anthraquinone intercalator conjugates, designed in such a way to allow their easy synthesis and modification, have been investigated as hydrolytic cleaving agents for plasmid DNA. The ligand structure comprises a triaminocyclohexane platform linked by means of alkyl spacers of different length (from C(4) to C(8)) to the anthraquinone group which may intercalate the DNA. At a concentration of 5 microM, the complex of the derivative with a C(8) alkyl spacer induces the hydrolytic stand scission of supercoiled DNA with a rate of 4.6 x 10(-6) s(-1) at pH 7 and 37 degrees C. The conjugation of the metal complex with the anthraquinone group leads to a 15-fold increase of the cleavage efficiency when compared with the anthraquinone lacking Zn-triaminocyclohexane complex. The straightforward synthetic procedure employed, allowing a systematic change of the spacer length, made possible to gain more insight on the role of the intercalating group in determining the reactivity of the systems. Comparison of the reactivity of the different complexes shows a remarkable increase of the DNA cleaving efficiency with the length of the spacer. In the case of too-short spacers, the advantages due to the increased DNA affinity are canceled due to the incorrect positioning of the reactive group, thus leading to cleavage inhibition.

Effects of calcium ions on the interactions between antithrombin and factor Xa mediated by variously sulfated, semisynthetic low-molecular-weight heparins.

A homogeneous set of low-molecular weight heparins, chemically modified to yield different degrees of sulfation, were investigated for their ability to interfere with the antithrombin (AT)-factor Xa (FXa) interaction process in the presence or absence of physiological concentrations of calcium ions. The heparin-AT dissociation constants were not appreciably affected by the presence of the metal ion, whereas the catalytic process was strongly dependent on Ca 2+. Our data suggest that AT binding to heparin represents the main factor driving the FXa inhibition process. In addition, the presence of the metal ion is likely to mask favorable AT- heparin ionic contacts occurring with the highly sulfated material. These results help in assessing proper structure-activity relationships for glycosaminoglycans, a multitarget family of biologically active compounds.