Transbuccal delivery of 5-Aza-2′-deoxycytidine: Effects of drug concentration,buffer solution,and bile salts on permeation

Delivery of 5-aza-2′-deoxycytidine (decitabine) across porcine buccal mucosa was evaluated as an alternative to the complex intravenous infusion regimen currently used to administer the drug. A reproducible high-performance liquid chromatography method was developed and optimized for the quantitative determination of this drug. Decitabine showed a concentration-dependent passive diffusion process across porcine buccal mucosa. An increase in the ionic strength of the phosphate buffer from 100 to 400 mM decreased the flux from 3.57±0.65 to 1.89±0.61 μg/h/cm². Trihydroxy bile salts significantly enhanced the flux of decitabine at a 100 mM concentration (P>.05). The steady-state flux of decitabine in the presence of 100 mM of sodium taurocholate and sodium glycocholate was 52.65±9.48 and 85.22±7.61 μg/cm²/h, respectively. Two dihydroxy bile salts, sodium deoxytaurocholate and sodium deoxyglycocholate, showed better enhancement effect than did trihydroxy bile salts. A 38-fold enhancement in flux was achieved with 10 mM of sodium deoxyglycocholate. Published: July 13, 2007     

HPLC method for determination of in vitro delivery through and into porcine skin of adefovir (PMEA)

A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10 mM KH2PO4 and 2 mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1-50 microg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6 microg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48 h were 294, 263, and 971 microg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates.     

Formulation and evaluation of ethosomes for transdermal delivery of lamivudine

The purpose of the present research was to investigate the mechanism for improved intercellular and intracellular drug delivery from ethosomes using visualization techniques and cell line study. Ethosomal formulations were prepared using lamivudine as model drug and characterized in vitro, ex vivo and in vivo. Transmission electron microscopy, scanning electron microscopy, and fluorescence microscopy were employed to determine the effect of ethosome on ultrastructure of skin. Cytotoxicity and cellular uptake of ethosome were determined using T-lymphoid cell line (MT-2). The optimized ethosomal formulation showed 25 times higher transdermal flux (68.4 +/- 3.5 microg/cm(2)/h) across the rat skin as compared with that of lamivudine solution (2.8 +/- 0.2 microg/cm(2)/h). Microscopic studies revealed that ethosomes influenced the ultrastructure of stratum corneum. Distinct regions with lamellar stacks derived from vesicles were observed in intercellular region of deeper skin layers. Results of cellular uptake study showed significantly higher intracellular uptake of ethosomes (85.7% +/- 4.5%) as compared with drug solution (24.9% +/- 1.9%). The results of the characterization studies indicate that lipid perturbation along with elasticity of ethosomes vesicles seems to be the main contributor for improved skin permeation.     

Liquid transport properties of porcine tracheal epithelium.

Because of its possible importance to the etiology of cystic fibrosis lung disease, the ion and water transport properties of tracheal epithelium were studied. Net liquid flux (J(V)) across porcine tracheal epithelium was measured in vitro using blue dextran as a volume probe. Luminal instillation of isosmotic sucrose solution (280 mM) induced a small net secretion of liquid (7.0 +/- 1.7 nl x cm(-2) x s(-1)), whereas luminal hyposmotic sucrose solutions (220 or 100 mM) induced substantial and significant (P < 0.05) liquid absorption (34.5 +/- 12 and 38.1 +/- 7.3 nl x cm(-2) x s(-1), respectively). When the luminal solution was normal (isosmotic) Krebs buffer, liquid was absorbed at 10.2 +/- 1.1 nl x cm(-2) x s(-1). Absorptive J(V) was abolished by 100 microM amiloride in the luminal solution and significantly reduced when the luminal solution was Na(+)-free Krebs solution. Absorptive J(V) was not significantly affected by 300 microM 5-nitro-2-(3-phenylpropylamino)benzoate or 100 microM diphenylamine-2-carboxylic acid, both cystic fibrosis transmembrane conductance regulator protein (CFTR) inhibitors, in the instillate but was significantly reduced by 60% when the luminal solution was Cl(-)-free Krebs solution. We conclude that water freely permeates porcine tracheal epithelium and that absorption of liquid is normally driven by active transcellular Na(+) transport and does not require the CFTR.     

Effect of bile salts on the plasma concentration of immunoreactive vasoactive intestinal polypeptide in man

The effects of bile salts on the release of immunoreactive vasoactive intestinal polypeptide (IR-VIP) were investigated in men using a specific radioimmunoassay. Plasma IR-VIP was determined after extraction by the acid-acetone method (recovery 75 +/- 5%). Oral administration of 400 mg sodium taurocholate caused a rise in plasma IR-VIP from 18.5 +/- 1.3 pmol/l to 31.1 +/- 2.1 pmol/l after 30 min and 39.0 +/- 1.7 pmol/l after 60 min and return to the initial value after 120 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma IR-VIP from a basal level of 14.5 +/- 1.5 pmol/l to 36.3 +/- 1.2 pmol/l after 60 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma IR-VIP from 11.9 +/- 1.1 pmol/l to 25.6 +/- 1.8 pmol/l after 30 min. Perifusion of 1 mM taurocholate stimulated release of IR-VIP from human duodenal mucosa into the perifusate. These results suggest that bile salts may participate, at least in part, in the release of IR-VIP from the gut.     

Bile salt alteration of ion transport across jejunal mucosa.

The effect of conjugated dihydroxy and trihydroxy bile salts on electrolyte transport across isolated rabbit jejunal mucosa was studied. Both taurochenodeoxycholic acid and taurocholic acid increased the short-circuit current (Isc) in bicarbonate-Ringer solution but not in a bicarbonate-free, chloride-free solution. Taurochenodeoxycholic acid was significantly more effective than taurocholic acid in increasing Isc. The presence of theophylline prevented the taurochenodeoxycholic acid- and taurocholic acid-induced increase in Isc. Transmural ion fluxes across jejunal mucosa demonstrated that 2 mM taurochenodeoxycholic acid decreased net Na+ absorption, increased net Cl- secretion and increased the residual flux (which probably represents HCO3- secretion). These studies support the hypothesis that cyclic AMP may be a mediator of intestinal electrolyte secretion.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.     

Formulation and Evaluation of Buccal Patches for Delivery of Atenolol

Buccal patches for the delivery of atenolol using sodium alginate with various hydrophilic polymers like carbopol 934 P, sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose in various proportions and combinations were fabricated by solvent casting technique. Various physicomechanical parameters like weight variation, thickness, folding endurance, drug content, moisture content, moisture absorption, and various ex vivo mucoadhesion parameters like mucoadhesive strength, force of adhesion, and bond strength were evaluated. An in vitro drug release study was designed, and it was carried out using commercial semipermeable membrane. All these fabricated patches were sustained for 24 h and obeyed first-order release kinetics. Ex vivo drug permeation study was also performed using porcine buccal mucosa, and various drug permeation parameters like flux and lag time were determined.     

Inhibition of active sodium transport by cytochalasin B in rat jejunum in vitro

The effects of cytochalasin B on electrophysiological properties and sodium transport in rat jejunum in vitro are described. Stripped paired rat jejunal segments were maintained in Ussing chambers with Leibovitz's (L-15) tissue culture medium bubbled with 100% oxygen. L-15 medium contains galactose as the only sugar, and an assortment of amino acids and cofactors to nourish the tissue. Electrophysiological parameters of short-circuit current (Isc) and transepithelial potential difference could be maintained for up to 4 h in control tissues. Upon application of cytochalasin B (20 micrograms/ml), on the mucosal side, Isc and potential difference fell within 1 h from 1.93 +/- 0.12 to 1.09 +/- 0.14 (mean +/- S.E.) muequiv./cm2 per h and from 5 to 2.5 mV. Tissue resistance remained unchanged at approx. 110 omega X cm2 for up to 4 h. 22Na net flux was 4.1 +/- 0.9 muequiv./cm2 per h during the last control period and fell to zero within 1 h after cytochalasin B treatment. Transmission electron micrographs revealed no gross morphological changes at this dose. Absorptive junctional morphology was apparently not altered by cytochalasin B treatment, a finding which was consistent with the stable transepithelial electrical resistance observed during exposure to this drug. Active sodium transport processes coupled to hexose, amino acid, and chloride movements are all possible in L-15 medium. However, following exposure to 20 micrograms/ml cytochalasin B, all net sodium transport is completely inhibited. The data are consistent with the hypothesis of a common regulator for active sodium transport processes which is modulated through structural changes in cytoskeletal organization.     

Effects of 16, 16-dimethyl prostaglandin E2 on bile-induced histamine release and permeability alterations in canine oxyntic mucosa.

Damage to the stomach results in excessive movement of hydrogen ion (H+) out of the lumen, and increased movement of sodium (Na+) and potassium (K+) into the lumen. Histamine liberation during damage probably adds to the destruction by increased capillary permeability and formation of edema. Previous reports have shown that the synthetic prostaglandin analogue 16,16-dimethyl prostaglandin E2 (Dm PGE2) protects dog gastric mucosa from aspirin- and ethanol-induced gastric mucosa damage. The effects of dm PGE2 on bile salt (sodium taurocholate) induced injury has not been investigated. Using the canine Heidenhain pouch, the present study examined the action of dm PGE2 on gastric mucosal damage induced by 5 mM sodium taurocholate in 100 mM HCl. Bile salt damaged the pouch mucosa as evidenced by an increased loss of H+, and increased net fluxes of both Na+ and K+. There was also an increase in the histamine content of the fluid irrigating the Heidenhain pouch. Intravenous injection of dm PGE2 in the doses 0.1 and 1.0 microgram/kg 1/2 h before administration of the sodium taurocholate in HCl significantly reduced the net loss of H+ and the gain of Na+, K+, and histamine. It is concluded the dm PGE2 effectively protects the canine gastric mucosa from the damaging effects of bile salt and that the mechanism of dm PGE2 protection of canine oxyntic mucosa may be mediated in part via inhibition of the gastric mucosal release of histamine.     

Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump

Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.     

Enhancing effects of bile salts on the degradation of glycosphingolipids by glycosidases from bacteria of the human fecal flora

Different concentrations of ionic and non-ionic detergents were examined for optimization of the in vitro degradations of intestinal glycosphingolipids by alpha- and beta-glycosidases from human fecal bacteria. In 5 mM Triton X-100 the enzymes hydrolyzed glycosphingolipids with lactoseries type 1 and 2 chains essentially to lactosylceramide (LacCer). In 5 mM sodium di- and trihydroxy bile salts lactosylceramide was degraded to glycosylceramide (GlcCer) in varying extent by enzymes from all five strains. The minimal bile salt concentrations for optimal 1,4-beta-galactosidase activities varied between 1 and 20 mM, i.e., close to or above the critical micellar concentrations (cmc). Dihydroxy bile salts were the most efficient in promoting conversion of LacCer to GlcCer at concentrations below 10 mM and conjugation with a taurine residue did not markedly lower the GlcCer yield. The optimal detergent concentrations for hydrolyses of the p-nitrophenyl (pnp) glycosides Gal beta 1-pnp and GalNAc alpha 1-pnp were approximately 0.05 mM for Triton X-100 and 0.5 mM for sodium taurodeoxycholate, i.e., clearly below their reported cmc values. Galabiosylceramide, globotria- and globotetraosylceramides, not degraded in the Triton X-100 micelles, were also resistant to hydrolysis using the sodium bile salts as detergents. In contrast, lactotetraosylceramide and isoglobotriaosylceramide were significantly more degraded by enzymes from a Ruminococcus gnavus strain and gangliotetraosylceramide by enzymes from a Bifidobacterium bifidum and a Bifidobacterium infantis strain using bile salt detergents. All strains but R. gnavus released terminal GalNAc from para-Forssman but not from the globotetraosylceramide or Forssman structures using 5 mM sodium deoxycholate as detergent. GM1 desialylation by two Ruminococcus torques strains and the R. gnavus and B. bifidum strains were enhanced under identical conditions. We conclude that the observed effects on glycosphingolipid hydrolyses reflects variations in the micellar presentation of the substrates. In addition, detergents seem to have a direct stimulating effect on the glycosidases, however at concentrations 10-100-times below the ones optimal for glycolipid degradations. These results with optimized bile salt concentrations, further support our previous observations that these five fecal bacterial strains produce enzymes with selected specificities towards glycosphingolipid core chains of the lactoseries type 1 and 2.     

Regulation of cytosolic sodium ion activity in frog sartorius

To explore the regulation of cytosolic sodium ion activity in the frog sartorius, we used Na(+)-selective microelectrodes to monitor intracellular sodium ion activity in situations of lowering external sodium concentration and elevating external potassium concentration. Reductions of 20%, 40%, 60% and 80% in extracellular sodium concentration produced slight but statistically insignificant changes in the membrane potential of the muscle. However, cytosolic sodium ion activity decreased significantly from 10.0 +/- 1.1 mM to 7.8 +/- 1.1 mM, 7.1 +/- 1.4 mM, 6.5 +/- 1.2 mM and 5.9 +/- 1.1 mM, respectively. In addition, elevation of the external potassium concentration from 2 mM to 12 mM, 32 mM and 62 mM caused respective stepwise depolarization of membrane potential from -87.2 +/- 1.6 mV to -62.4 +/- 3.6 mV, -45.4 +/- 3.0 mV, -27.2 +/- 1.8 mV. Under these conditions, the cytosolic sodium ion activity decreased from 10.5 +/- 1.4 mM to 7.3 +/- 1.6 mM, 6.4 +/- 1.1 mM and 5.2 +/- 0.8 mM, respectively. The results illustrate that the net sodium flux is out of cell either in the reduction of sodium chemical gradient or in the potassium depolarization across the cell membrane.     

Rat intestinal ceramidase: purification, properties, and physiological relevance

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.     

Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase

Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the V(max) was 930 micromol/h/mg and K(m) was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more effective than glycine conjugated ones, and the most effective bile salts were taurocholate and taurochenodeoxycholate. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X100 (TX100) had no stimulatory effects. Unlike neutral SMase, intestinal alkaline SMase was not Mg2+ dependent, not inhibited by EDTA, and not inhibited by glutathione. The enzyme was stable during incubation with temperatures up to 50 degree C and in pHs from 7 to 10. Trypsin and chymotrypsin had no effects on its activity, and 10 mM dithiothreitol reduced its activity by 25%. A specific antibody against the enzyme was developed, and Western blot showed that the enzyme was expressed in the intestine but not in other organs. In conclusion, we purified a potentially important SMase in the intestine with several properties different from neutral SMase.     

In vivo percutaneous absorption of boron as boric acid, borax, and disodium octaborate tetrahydrate in humans: a summary

Literature from the first half of this century reports concern for toxicity from topical use of boric acid, but assessment of percutaneous absorption has been impaired by lack of analytical sensitivity. Analytical methods in this study included inductively coupled plasma-mass spectrometry, which now allows quantitation of percutaneous absorption of 10B in 10B-enriched boric acid, borax, and disodium octaborate tetrahydrate (DOT) in biological matrices. This made it possible, in the presence of comparatively large natural dietary boron intakes for the in vivo segment of this study, to quantify the boron passing through skin. Human volunteers were dosed with 10B-enriched boric acid, 5.0%, borax, 5.0%, or disodium octaborate tetrahydrate, 10% in aqueous solutions. Urinalysis, for boron and changes in boron isotope ratios, was used to measure absorption. Boric acid in vivo percutaneous absorption was 0.226 (SD = 0.125) mean percent dose, with flux and permeability constant (Kp) calculated at 0.009 microg/cm2/h and 1.9 x 10(-7) cm/h, respectively. Borax absorption was 0.210 (SD = 0.194) mean percent dose, with flux and Kp calculated at 0.009 microg/cm2/h and 1.8 x 10(-7) cm/h, respectively. DOT absorption was 0.122 (SD = 0.108) mean percent, with flux and Kp calculated at 0.01 microg/cm2/h and 1.0 x 10(-7) cm/h, respectively. Pretreatment with the potential skin irritant 2% sodium lauryl sulfate had no effect on boron skin absorption. These in vivo results show that percutaneous absorption of boron, as boric acid, borax, and disodium octaborate tetrahydrate, through intact human skin is low and is significantly less than the average daily dietary intake. This very low boron skin absorption makes it apparent that, for the borates tested, the use of gloves to prevent systemic uptake is unnecessary. These findings do not apply to abraded or otherwise damaged skin.     

Mutagenicity studies on paracetamol in human volunteers. II. Unscheduled DNA synthesis and micronucleus test

The possible genotoxic effect of paracetamol (PC) was studied in a group of 11 healthy volunteers. PC was administered in the form of tablets 3 x 1000 mg in the course of 8 h. Blood samples and buccal mucosa cells were taken 0, 24, 72 and 168 h after the first administration of the drug. Each blood sample was used for the termination of the unscheduled DNA synthesis (UDS) in peripheral lymphocytes and ascorbemia in plasma. Buccal mucosa cells were analysed for micronuclei. After PC administration the level of UDS induced by MNNG was decreased to T/C = 4.11 +/- 0.56 after 24 h vs. T/C = 5.02 +/- 0.47 (p less than 0.01) at 0 h. The frequency of micronucleated cells in the buccal mucosa was increased after 72 h to 0.38 +/- 0.07% vs. 0.19 +/- 0.06% (p less than 0.01) before PC administration. If PC was administered simultaneously with ascorbic acid (AA), also in a dose of 3 X 1000 mg, a decreased level of UDS was observed after 24, 72 and 168 h and the increased number of micronuclei was qualitatively the same as the PC alone: 0.38 +/- 0.09% after 72 h vs. 0.20 +/- 0.05% at 0 h AA did not decrease the genotoxic effect of PC, but prolonged the influence of PC on UDS.     

Mechanisms of diarrhea in the interleukin-2-deficient mouse model of colonic inflammation

Colitis in interleukin-2-deficient (IL-2(-/-)) mice resembles ulcerative colitis in humans. We studied epithelial transport and barrier function in IL-2(-/-) mice and used this model to characterize mechanisms of diarrhea during intestinal inflammation. (22)Na(+) and (36)Cl(-) fluxes were measured in proximal colon. Net Na(+) flux was reduced from 4.0 +/- 0.5 to 0.8 +/- 0.5 micromol.h(-1).cm(-2), which was paralleled by diminished mRNA and protein expression of the Na(+)/H(+) exchanger NHE3. Net Cl(-) flux was also decreased from 2.2 +/- 1.6 to -2.7 +/- 0.6 micromol.h(-1).cm(-2), indicating impaired Na(+)-Cl(-) absorption. In distal colon, aldosterone-induced electrogenic Na(+) absorption was 6.1 +/- 0.9 micromol.h(-1).cm(-2) in controls and was abolished in IL-2(-/-) mice. Concomitantly, mRNA expression of beta- and gamma-subunits of the epithelial sodium channel (ENaC) was reduced. Epithelial barrier was studied in proximal colon by impedance technique and mannitol fluxes. In contrast to ulcerative colitis, epithelial resistance was increased and mannitol fluxes were decreased in IL-2(-/-) mice. This was in accord with the findings of reduced ion transport as well as increased expression of tight junction proteins occludin and claudin-1, -2, -3, and -5. In conclusion, the IL-2(-/-) mucosa exhibits impaired electroneutral Na(+)-Cl(-) absorption and electrogenic Na(+) transport due to reduced mRNA and protein expression of NHE3 and ENaC beta- and gamma-subunit mRNA. This represents a model of early intestinal inflammation with absorptive dysfunction due to impaired transport protein expression/function while epithelial barrier is still intact. Therefore, this model is ideal to study regulation of transporter expression independent of barrier defects.     

Embryotrophic effects of ethylenediaminetetraacetic acid and hemoglobin on in vitro porcine embryos development

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.     

Intestinal zinc transport: influence of streptozotocin-induced diabetes, insulin and arachidonic acid

The influence of arachidonic acid (AA) on the zinc flux rates of jejunal segments, isolated from streptozotocin-induced diabetic rats injected with saline or with insulin, was investigated using an Ussing chamber technique. Although the zinc flux rates from mucosa-to-serosa (Jms) of normal rats were inhibited by addition of 5 microM AA to the jejunal segment bathing medium (46.4 +/- 5.0 vs 32.6 +/- 4.3 nmol/hr/cm2), AA had no effect on the Jms of diabetic rats either with or without insulin treatment. Induction of diabetes also significantly reduced Jms (46.4 +/- 5.0 vs 22.1 +/- 4.9 nmol/hr/cm2), but 3 day insulin treatment (NPH 8 U/Kg/day subcutaneously) did not reverse this effect (29.2 +/- 5.1 nmol/hr/cm2). Addition of AA to the serosal side did not significantly alter the zinc flux rate from serosa-to-mucosa (Jsm) in either control, diabetic or diabetic rats treated with insulin. The net zinc absorption rate (Jnet) of jejunal segments was decreased in diabetic rats compared to controls (13.2 +/- 3.0 vs -0.7 +/- 2.1 nmol/hr/cm2), but normalization of blood glucose with 3 day insulin treatment did not increase Jnet. Addition of AA was associated with a tendency to increase zinc uptake capacity. This change reached statistical significance in insulin treated diabetic rats. Short-circuit current (Isc) for diabetic rats was increased compared to controls but addition of AA to the mucosal side bathing medium decreased Isc in all groups. The results indicate that the zinc flux rate in the small intestine of streptozotocin-induced diabetic rats is decreased, that zinc uptake capacity of the small intestine does not directly reflect the zinc flux rate across the small intestine, and that AA or one of its metabolites may play a significant role in the control of the zinc flux across the intestinal epithelium.