23 research outputs found
BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium
Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1âĂâ1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41â±â3.17â
mm; AdLacZ: 3.07â±â1.33â
mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD
Designing the Ergonomic Properties of Pruning Shears Using Quality Function Deployment (QFD)
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 6\u3b2-hydroxylation; one CYP3A38 variant increased TST 16\u3b2-hydroxylation, while a CYP3A48 SNV showed enhanced NIF oxidation. Individuals homozygous for rs384467435 SNV showed a reduced TST 6\u3b2-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
New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis
Metabolism of bilirubin by human cytochrome P450 2A6
The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic "Bilirubin Oxidase" (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K-i of 2.231 mu M. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301,315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human "Bilirubin Oxidase" where bilirubin is potentially a substrate and a regulator of the enzyme. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved
<i>In Silico</i> Prediction of the Site of Oxidation by Cytochrome P450 3A4 That Leads to the Formation of the Toxic Metabolites of Pyrrolizidine Alkaloids
In
humans, the metabolic bioactivation of pyrrolizidine alkaloids
(PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation
of their necine bases at C3 or C8 of heliotridine- and retronecine-type
PAs or at the N atom of the methyl substituent of otonecine-type PAs.
However, no attempts have been made to identify which C atom is the
most favorable site for hydroxylation <i>in silico</i>.
Here, in order to determine the site of hydroxylation that eventually
leads to the formation of the toxic metabolites produced from lasiocarpine,
retrorsine, and senkirkin, we utilized the ligand-based electrophilic
Fukui function <i>f</i><sup>â</sup>(<b>r</b>) and hydrogen-bond dissociation energies (BDEs) as well as structure-based
molecular docking. The ligand-based computations revealed that the
C3 and C8 atoms of lasiocarpine and retrorsine and the C26 atom of
senkirkin were chemically the most susceptible locations for electrophilic
oxidizing reactions. Similarly, according to the predicted binding
orientation in the active site of the crystal structure of human CYP3A4
(PDB code: 4I4G), the alkaloids were positioned in such a way that the C3 atom of
lasiocarpine and retrorsine and the C26 of senkirkin were closest
to the catalytic heme Fe. Thus, it is concluded that the C3 atom of
lasiocarpine and retrorsine and C26 of senkirkin are the most favored
sites of hydroxylation that lead to the production of their toxic
metabolites
Identification of a New Reactive Metabolite of Pyrrolizidine Alkaloid Retrorsine: (3<i>H</i>âPyrrolizin-7-yl)methanol
Pyrrolizidine alkaloids (PAs) such
as retrorsine are common food
contaminants that are known to be bioactivated by cytochrome P450
enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites
known as dehydropyrrolizidine alkaloids (DHPs). We compared how both
electrochemical (EC) and human liver microsomal (HLM) oxidation of
retrorsine could produce short-lived intermediate metabolites; we
also characterized a toxicologically important metabolite, (3<i>H</i>-pyrrolizin-7-yl)Âmethanol. The EC cell was coupled online
or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas
the HLM oxidation was performed in 100 mM potassium phosphate (pH
7.4) in the presence of NADPH at 37 °C. The EC cell oxidation
of retrorsine produced 12 metabolites, including dehydroretrorsine
(<i>m</i>/<i>z</i> 350, [M + H<sup>+</sup>]),
which was degraded to a new reactive metabolite at <i>m</i>/<i>z</i> 136 ([M + H<sup>+</sup>]). The molecular structure
of this small metabolite was determined using high-resolution mass
spectrometry and NMR spectroscopy followed by chemical synthesis.
In addition, we also identified another minor but reactive metabolite
at <i>m</i>/<i>z</i> 136, an isomer of (3<i>H</i>-pyrrolizin-7-yl)Âmethanol. Both (3<i>H</i>-pyrrolizin-7-yl)Âmethanol
and its minor isomer were also observed after HLM oxidation of retrorsine
and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the
presence of reduced glutathione (GSH), each isomer formed identical
GSH conjugates at <i>m</i>/<i>z</i> 441 and <i>m</i>/<i>z</i> 730 in the negative ESI-MS. Because
(3<i>H</i>-pyrrolizine-7-yl)Âmethanol) and its minor isomer
subsequently reacted with GSH, it is concluded that (3<i>H</i>-pyrrolizin-7-yl)Âmethanol may be a common toxic metabolite arising
from PAs