Assessment of developmental toxicity of vorinostat, a histone deacetylase inhibitor, in Sprague-Dawley rats and Dutch Belted rabbits

BACKGROUND: The developmental toxicity potential of vorinostat (suberoylanilide hydroxamic acid [SAHA], ZOLINZA), a potent inhibitor of histone deacetylase (HDAC), was assessed in Sprague-Dawley rats and Dutch Belted rabbits. HDAC inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation, and apoptosis of tumor cells. Range-finding studies established oral dose levels of 5, 15, or 50 mg/kg/day and 20, 50, or 150 mg/kg/day in rats and rabbits, respectively. METHODS: Animals were dosed on Gestation Days 6-20 or 7-20, respectively, with litter/fetal parameters evaluated on GD 21 and 28, respectively. Separate studies evaluated toxicokinetic parameters at the mid- and high-dose levels. RESULTS: There was no maternal toxicity observed at the highest dose levels; however, hematology and serum biochemistry changes were characterized in the range-finding studies. Vorinostat did not induce morphological malformations in either rat or rabbit fetuses. In rats, drug-related developmental toxicity was observed only in the high-dose group and consisted of markedly decreased fetal weight and increases in fetuses with a limited number of skeletal variations. In rabbits, drug-related developmental toxicity was also observed only in the high-dose group and consisted of slightly decreased fetal weight and increases in fetuses with a short 13th rib and incomplete ossification of metacarpals. Maternal exposures to vorinostat based on AUC and Cmax values were comparable at the high-dose levels of both species. Rabbits tolerated higher dosages probably due to more extensive metabolism. Maternal concentrations of vorinostat were approximately 1,000-fold above the known in vitro HDAC inhibitory concentration. CONCLUSIONS: Review of previous work with valproic acid, another HDAC inhibitor, suggest that the developmental toxicity profiles of these 2 compounds are not the result of HDAC inhibition but involve other mechanisms.     

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.     

Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.     

Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution +/- glutathione (GSH) or +/- polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 degrees C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 +/- 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 +/- 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 degrees C) in the presence of iron (160 microM) MDA formation was maximally stimulated (3.314 +/- 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA-Seq vs Dual- and Single-Channel Microarray Data: Sensitivity Analysis for Differential Expression and Clustering

With the fast development of high-throughput sequencing technologies, a new generation of genome-wide gene expression measurements is under way. This is based on mRNA sequencing (RNA-seq), which complements the already mature technology of microarrays, and is expected to overcome some of the latter’s disadvantages. These RNA-seq data pose new challenges, however, as strengths and weaknesses have yet to be fully identified. Ideally, Next (or Second) Generation Sequencing measures can be integrated for more comprehensive gene expression investigation to facilitate analysis of whole regulatory networks. At present, however, the nature of these data is not very well understood. In this paper we study three alternative gene expression time series datasets for the Drosophila melanogaster embryo development, in order to compare three measurement techniques: RNA-seq, single-channel and dual-channel microarrays. The aim is to study the state of the art for the three technologies, with a view of assessing overlapping features, data compatibility and integration potential, in the context of time series measurements. This involves using established tools for each of the three different technologies, and technical and biological replicates (for RNA-seq and microarrays, respectively), due to the limited availability of biological RNA-seq replicates for time series data. The approach consists of a sensitivity analysis for differential expression and clustering. In general, the RNA-seq dataset displayed highest sensitivity to differential expression. The single-channel data performed similarly for the differentially expressed genes common to gene sets considered. Cluster analysis was used to identify different features of the gene space for the three datasets, with higher similarities found for the RNA-seq and single-channel microarray dataset.     

Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance

Background

Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial ‘set-point’ may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the ‘reference range of normality’ remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport.

Methodology/Principal Findings

Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm²), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm²; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport.

Conclusions/Significance

Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine.     

Reduction of chronic hypoxic pulmonary hypertension in the rat by beta-aminopropionitrile

We administered antifibrotic agent beta-aminopropionitrile (BAPN) to rats exposed to 10% O2-90% N2 for 3 wk to prevent excess vascular collagen accumulation. Groups of Sprague-Dawley rats studied were air breathing, hypoxic, and hypoxic treated with BAPN, 150 mg/kg twice daily intraperitoneally. After the 3-wk period, we measured mean right ventricular pressure (RVP), the ratio of weight of right ventricle to left ventricle plus septum (RV/LV + S), and hydroxyproline content of the main pulmonary artery (PA) trunk. Hypoxia increased RVP from 14 to 29 mmHg; RVP was 21 mmHg in hypoxic BAPN-treated animals. Hypoxia increased the RV/LV + S ratio from 0.28 to 0.41; the ratio was 0.32 in hypoxic BAPN-treated animals. Hypoxia increased PA hydroxyproline from 20 to 239 micrograms/artery; hydroxyproline was 179 micrograms/artery in hypoxic BAPN-treated animals. Thus BAPN prevented pulmonary hypertension, right ventricular hypertrophy, and excess vascular collagen produced by hypoxia. We conclude that vascular collagen contributes to the maintenance of chronic hypoxic pulmonary hypertension.