Xiaoda Yang

Studies of intestinal permeability of 36 flavonoids using Caco-2 cell monolayer model.

To investigate the structure-permeability relationship of dietary/nutriceutic flavonoids, the transepithelial transport and cellular uptake of 36 flavonoids (including flavones, flavonols, dihydroflavones, dihydroflavonols, isoflavones, chalcones, flavanes, flavanols, methylated and glycosidic derivatives) were investigated using the Caco-2 cell monolayer. The apparent permeability coefficients (P(app)) of the flavonoids were calculated from bilateral transport assays in the Transwell system with flavonoid determination using a high performance liquid chromatography (HPLC) coupled with a UV detector. The most flavonoids exhibited concentration-independent P(app) values and a ratio of 0.5-1.8 for P(app AB to BL)/P(app BL to AB), suggesting passive diffusion pathways. However, certain flavonoids e.g. morin and some flavonoid glycosides may involve the efflux mechanisms. For isoflavones, flavones, and dihydroflavones, the oil/water partition coefficients (additionally modified by the number and position of the three hydroxyl groups) was the key determinant for Caco-2 cell permeation. However, the permeability of flavonols is more complex with their structure possibly related to their high rate of cell accumulation. Overall, the parental skeleton structure, the number and position of free hydroxyl groups, accumulation and efflux in Caco-2 cell play the key roles in the transport of flavonoids across Caco-2 cell monolayer.

Vanadium compounds induced mitochondria permeability transition pore (PTP) opening related to oxidative stress

Vanadium compounds have been regarded as promising in therapeutic treatment of diabetes and in cancer prevention. In the present work, we studied the effects of vanadium compounds on mitochondria to investigate the mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with a variety of vanadium compounds, i.e. VOSO(4), NaVO(3), and vanadyl complexes with organic ligands. Our studies indicated that VO(2+), VO(3)(-), VO(acac)(2) and VOcit (1-100microM) could induce mitochondrial swelling in a concentration dependent manner and disrupt mitochondrial membrane potential (Deltapsi(m)) in a time dependent manner, which is quite different from the rapid Deltapsi(m) collapse caused by Ca(2+) or CCCP (carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupling reagent). Release of cytochrome c (Cyt c) was observed and could be inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (PTP). Interestingly, VOdipic caused release of Cyt c without mitochondrial swelling and Deltapsi(m) disruption, an action previously only observed on the Bax protein, suggesting a potentially role of VOdipic in regulating PTP opening. In addition, all the vanadium compounds tested stimulated mitochondrial production of reactive oxygen species (ROS). Antioxidants, i.e. vitamin C and E, significantly delayed the Deltapsi(m) disruption. Overall, our experimental evidence indicated vanadium compounds exhibited multiple actions on mitochondria. Vanadium compounds did induce oxidative stress on mitochondrial and thus caused PTP opening, which led to collapse of Deltapsi(m) and Cyt c release as the initiation of cell apoptosis.

The permeability and cytotoxicity of insulin-mimetic vanadium compounds.

AbstractPurpose. The aim of this study was to investigate the mechanism of permeation and cytotoxicity of vanadium compounds, [VO(acac)2], [VO(ma)2], and vanadate. Methods. Absorptive transport were carried out in Caco-2 monolayers grown on transwell inserts. Vanadium was quantified using inductively coupled plasma atomic emission spectrometry (ICP-AES). The change of Caco-2 cells in the microvilli morphology and F-actin structure was visualized by transmission electron microscopy and confocal laser scanning microscopy. Results. The three vanadium compounds were taken up by Caco-2 cells via simple passive diffusion. [VO(acac)2] were mainly transcellularly transported and exhibited the highest apparent permeabilty coefficients (8.2 × 10-6 cm-1). The cell accumulation of [VO(acac)2] was found to be greater than that of [VO(ma)2], and vanadate caused much less accumulation than the other two compounds. Vanadium compounds induced intracellular reactive oxygen species, reduced the transepithelial electric resistance, caused morphological change in microvilli, and led to different perturbation of F-actin structure. Conclusions. The three compounds exhibited different permeability due to different diffusion process and cellular uptake. The toxicity of vanadium complexes on Caco-2 monolayer involved F-actin-related change of tight junction and impairment of microvilli. The toxicity was also related to elevated intracellular reactive oxygen species (ROS) and their cellular accumulation.

La3+, Gd3+ and Yb3+ induced changes in mitochondrial structure, membrane permeability, cytochrome c release and intracellular ROS level

Lanthanides (Ln) were known to induce cell apoptosis, which might be the results of their effects on mitochondria (MT). This study was trying to clarify the role of MT and reactive oxygen species (ROS) in Ln-induced apoptosis. We found that micromolar or lower concentration of La(3+), Gd(3+) and Yb(3+) bound to MT and induced swelling of isolated MT; EGTA treatment can inhibit the process. In addition, La(3+), Gd(3+) and Yb(3+) increased the MT membrane fluidity and decreased the MT membrane potential (DeltaPsi(m)). All these were inferred to the results of MT permeability transition pore opening. Release of cytochrome c (Cyt-c) from the MT upon incubation with Ln ions was monitored by immunocytochemistry, however, Cyt-c release was observed only in the cytosol of cells. In parallel with these events, there was a higher level of ROS found in the cells exposed to Ln. It was proposed that Ln-induced apoptosis via the MT pathways and it was highly possible that ROS were involved in the mechanism.

Insulin-enhancing activity of a dinuclear vanadium complex: 5-chloro-salicylaldhyde ethylenediamine oxovanadium(V) and its permeability and cytotoxicity

A new insulin-enhancing oxovanadium complex 5-chloro-salicylaldhyde ethylenediamine oxovanadium (V) ([V(2)O(2)(mu-O)(2)L(2)]) has been synthesized. The complex was characterized by a variety of physical methods, including X-ray crystallography. The X-ray diffraction analysis show a dinuclear complex of two six-coordinate vanadium centers doubly bridged by the oxygen atoms of the Schiff base ligand with a V(2)O(2) diamond core. The complex was administered intragastrically to STZ-diabetic rats for 2 weeks. The biological activity results show that the complex at the dose of 10.0 and 20.0 mg V kg(-1), could significantly decrease the blood glucose level and ameliorate impaired glucose tolerance in STZ-diabetic rats. That results suggested that the complex exerts an antidiabetic effect in STZ-diabetic rats. Furthermore, the complex ([V(2)O(2)(mu-O)(2)L(2)]) had permeability above 10(-5)cm/s. The experimental results suggested that the vanadium complex permeates via a passive diffusion mechanism. It was also suggested the complex with salen-type ligands has good lipophilic properties and better oral administration. The cytotoxicity of the complex ([V(2)O(2)(mu-O)(2)L(2)]) on Caco-2 cells was measured by a decrease of cell viability using the MTT assay suggesting that the chlorine atom at C4 of complex [V(2)O(2)(mu-O)(2)L(2)] increased cytotoxicity for vanadium complexes.

Impaired mitochondrial function and oxidative stress in rat cortical neurons: implications for gadolinium-induced neurotoxicity.

Gadolinium (Gd), a rare-earth lanthanides metal, is widely utilized for various industrial and medical purposes, particularly in brain magnetic resonance imaging. However, its potential effects on the impairment of the central nervous system remain uncertain, especially with regard to the mitochondria, the potential primary target in metal-induced neural injury. This study investigates the effects of gadolinium on mitochondrial energy metabolism, ROS accumulation, and cell death toward cortical neurons. Results show that the metabolic activity of the mitochondria significantly decreased as early as 3h after exposure of cells to gadolinium chloride. Subsequently, significant elevation of intracellular ROS, decrease in ATP synthesis, depolarization of mitochondrial membrane potential, release of cytochrome c and activation of caspase-3 were observed. Following these changes, increased release of LDH into culture medium and DNA fragmentation were detected. Inhibition of both cytochrome c release and caspase-3 activation could significantly reduce Gd-induced neuron cell death. All these results suggest that gadolinium cause neuron cell apoptosis primarily by inhibiting mitochondrial function and inducing oxidative stress. The present work provides new insight into the toxicological mechanism of gadolinium in neurons.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection.

Gadolinium-induced oxidative stress triggers endoplasmic reticulum stress in rat cortical neurons

J. Neurochem. (2011) 117, 38–47.

Bioprocess of uniform-sized crosslinked chitosan microspheres in rats following oral administration

Chitosan microspheres have a great potential in pharmaceutical application. In this study, uniform-sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) were prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. Based on the characterizations of uniform size and autofluorescence, it was possible to develop a new detecting system for observing and quantifying the CG microspheres in rats with three different diameters (2.1, 7.2 and 12.5 microm) synchronously after oral administration. This system was a combination of scanning electron microscopy (SEM), laser scanning confocal microscope (LSCM) and flow cytometer technique, which showed the advantages of being simple, intuitionistic, repeatable and sensitive. After oral administration of three kinds of particles with different diameters, bioadhesion in gastrointestinal tract, absorption in gastrointestinal tract, distribution in systemic tissues, and biodegradation in reticuloendothelial system (RES) were studied firstly in detail. The CG microspheres showed different fates in bioadhesion, absorption and distribution according to their diameters, while the biodegradation also varied due to the different locations in RES. These original results would indicate a better way for the CG microspheres in the clinical application.

A new salicylic acid-derivatized kojic acid vanadyl complex: synthesis, characterization and anti-diabetic therapeutic potential.

The molecular mechanisms of vanadium toxicity suggest that incorporation of antioxidant groups in the structure of vanadium complexes could be a preferable strategy in designing novel hypoglycemic vanadium complexes with proper efficacy and safety. By conjugating a pyrone skeleton to provide a coordination group and antioxidative motifs, we synthesized a novel complex of bis ((5-hydroxy-4-oxo-4H-pyran-2-yl) methyl 2-hydroxy- benzoatato) oxovanadium (IV) (BSOV). Evaluation of the anti-diabetic effects of BSOV using streptozotocin (STZ)-induced diabetic rats with bis (maltolato) oxovanadium (BMOV) as a positive control showed that BSOV effectively lowered blood glucose level, ameliorated damages of hepatic and renal function in diabetic rats and improved lipid metabolism. The signs of potential alteration of renal function caused by BSOV and BMOV were observed and are discussed. Overall, the experimental results suggest BSOV as a potent hypoglycemic agent and further studies using this strategy for anti-diabetic agents.