Effect of bisphenol A on drug efflux in BeWo, a human trophoblast-like cell line.

Bisphenol A (BPA) is a monomer of polycarbonate plastics that has estrogenic activities and has been shown to be a substrate for multidrug resistant efflux mechanisms, specifically, P-glycoprotein. Since the natural hormone estrogen reverses multidrug resistance in some cell types, we hypothesized that BPA might have a similar activity in trophoblasts. We have used BeWo cells as an in vitro model for human trophoblasts and calcein AM as a substrate for drug efflux mechanism to characterize BPA interactions with placental P-glycoprotein. We found that chronic exposure of BeWo cells to BPA did not alter intracellular calcein accumulation in a fashion that would be reflective of changes in P-glycoprotein expression. Immunoblots affirmed that BPA had small effects on P-glycoprotein expression. However, BeWo cells acutely exposed to BPA pretreatment were observed to have a significantly decreased calcein accumulation. Addition of cyclosporin A, a P-glycoprotein inhibitor and substrate, completely reversed BPA's effects on calcein accumulation and resulted in a net increase, relative to controls, in calcein accumulation by the BeWo cells. BPA was found not to stimulate P-gp ATPase or alter intracellular esterases mediating calcein release from calcein AM. Therefore, our results suggested that BPA stimulated drug efflux by BeWo cells probably by direct effects on P-glycoprotein.

{beta}-Amyloid-induced neurodegeneration and protection by structurally diverse microtubule-stabilizing agents.

Deposition of beta-amyloid peptide (Abeta) and hyperphosphorylation of the tau protein are associated with neuronal dysfunction and cell death in Alzheimer's disease. Although the relationship between these two processes is not yet understood, studies have shown that both in vitro and in vivo exposure of neurons to Abeta leads to tau hyperphosphorylation and neuronal dystrophy. We previously reported that the microtubule-stabilizing drug paclitaxel (Taxol) protects primary neurons against toxicity induced by the Abeta(25-35) peptide. The studies in this report were undertaken to characterize the actions of paclitaxel more fully, to assess the effectiveness of structurally diverse microtubulestabilizing agents in protecting neurons, and to determine the time course of the protective effects of the drugs. Primary neurons were exposed to Abeta in the presence or absence of several agents shown to interact with microtubules, and neuronal survival was monitored. Paclitaxel protected neurons against Abeta(1-42) toxicity, and paclitaxel-treated cultures exposed to Abeta showed enhanced survival over Abeta-only cultures for several days. Neuronal apoptosis induced by Abeta was blocked by paclitaxel. Other taxanes and three structurally diverse microtubule-stabilizing compounds also significantly increased survival of Abeta-treated cultures. At concentrations below 100 nM, the drugs that protected the neurons did not produce detectable toxicity when added to the cultures alone. Although multiple mechanisms are likely to contribute to the neuronal cell death induced by oligomeric or fibrillar forms of Abeta, low concentrations of drugs that preserve the integrity of the cytoskeletal network may help neurons survive the toxic cascades initiated by these peptides.

The presence of inducible cytochrome P450 types 1A1 and 1A2 in the BeWo cell line.

The activity and inducibility of cytochrome P450 systems (CYP1A1:1A2) of the human placenta were assessed in a representative human trophoblast-like cell line, BeWo. The activity of CYP1A1 and CYP1A2 in microsome preparations from human liver, placenta, primary cultures of human cytotrophoblast, and BeWo cells was measured by O -dealkylation of 7-ethoxyresorufin (EROD) and 7-methoxyresorufin O -demethylation (MROD), respectively. Results indicated high EROD and MROD activity associated with human liver microsomes, sometimes comparable activities in human placenta microsomes prepared from smokers, and relatively low activities in human placenta microsomes from nonsmokers and in the primary cultures of cytotrophoblasts isolated from nonsmokers. Microsomes from BeWo cell monolayers exhibited the lowest EROD and MROD activities relative to all other microsome preparations. However, compared to primary cultures of normal trophoblasts, the EROD activity of the BeWo cells was far more sensitive to typical inducers, 3-methylcholanthrene, 1,2-benzanthracene, and beta-naphthoflavone. EROD activity in BeWo cells was induced approximately 200-fold by 3-methylcholanthrene. Both EROD and MROD activity in BeWo cells was readily induced by 1,2-benzanthracene, 100-fold and 60-fold, respectively. After induction with 1,2-benzanthracene, the CYP1A1 selective inhibitor, alpha-naphthoflavone, and the CYP1A2 selective inhibitor, furafylline, effectively inhibited enzyme activities with IC(50)s of 2.4 microM and 12.8 microM, respectively, in microsomes from both trophoblasts culture systems. These results show that major cytochrome P450 forms present in human placenta are present and inducible in BeWo cells, a potential model for investigation of drug metabolism mechanisms in the human trophoblast.

Carrier-mediated transport of folic acid in BeWo cell monolayers as a model of the human trophoblast.

Using cultured BeWo cells as a model of human trophoblast, we investigated whether carrier-mediated transport of folic acid occurs. BeWo cells, which were derived from human choriocarcinoma, were cultured on a tissue culture plate or in a permeation chamber. When the cells reached confluence, drug uptake or transport experiments were performed. The uptake of [(3)H]folic acid by BeWo cells occurred at a much lower rate at 4 degrees C than at 37 degrees C. The uptake of [(3)H]folic acid was saturable at higher concentrations and inhibited by typical metabolic inhibitors, sodium azide and 2,4-dinitrophenol. The uptake of [(3)H]folic acid was significantly increased with decreasing pH of the incubation buffer and markedly inhibited by 4,4'-diidothiocyanostilbene-2,2'-disulfonic acid (DIDS). Analogs of folic acid, methotrexate and 5-methyltetrahydrofolate, inhibited the uptake of [(3)H]folic acid by BeWo cells. Kinetic analysis using Lineweaver-Burk plots revealed that methotrexate competitively inhibited the uptake of [(3)H]folic acid and folic acid competitively inhibited the uptake of [(3)H]methotrexate. In transport experiments, the permeation of [(3)H]folic acid from the apical-to-basal side was greater than that from the basal-to-apical side, and the transport of [(3)H]folic acid from the apical-to-basal side was inhibited by an excess of folic acid. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for folic acid and its analog, methotrexate, across BeWo cells, a representative of the human trophoblast.

Modulation of P-glycoprotein activity in Calu-3 cells using steroids and beta-ligands.

The purpose of this work was to investigate if P-glycoprotein (Pgp) efflux pump activity could be inhibited in the sub-bronchial epithelial cell line, Calu-3, by glucocorticosteroids and beta-ligands. The Pgp modulation efficiency of each compound was determined by its ability to increase the accumulation of the Pgp substrate rhodamine 123 (Rh123) accumulation in these cells. Pgp inhibition was observed at > or =100 microM steroids and beta-ligand. The modulation effectiveness of the beta-ligands increased with increasing hydrophobicity (logP(octanol/aqueous)) whereas an obvious correlation was not obtained with the complete set of steroids tested. Steroidal Pgp substrates did not affect Rh123 accumulation (e.g. aldosterone, dexamethasone, 11beta,17alpha,21-OH progesterone). In contrast, two hydrophobic non-Pgp steroidal substrates (testosterone and progesterone) displayed different effects on Rh123 accumulation, with progesterone being the more potent modulator. The most hydrophobic beta-ligand, propranolol, a known Pgp substrate, gave the largest increase in Rh123 accumulation in this therapeutic class. The beta-ligand modulation efficiency could also be correlated to Pgp structural recognition elements such as hydrogen bonding potential, the presence of a basic nitrogen and planar aromatic ring. No effect on Rh123 accumulation was observed with the formulation additives tested (ethanol, glycerol and palmitoyl carnitine) at concentrations previously reported to be non-toxic to Calu-3 cells.

Multidrug resistance-associated protein-1 functional activity in Calu-3 cells.

The purpose of this work was to determine whether the in vitro bronchiolar epithelial cell model, Calu-3, possesses efflux pump activity by the multidrug resistance-associated protein-1 (MRP1). Reverse transcription-polymerase chain reaction demonstrated MRP1 gene expression in Calu-3 cells. Indirect fluorescence studies showed a basolateral membrane localization of MRP1 compared with P-glycoprotein (Pgp) that was found on the apical side of these cells. An increase in the rate of accumulation of the MRP1 substrate calcein was observed following treatment with the organic anion/MRP1 inhibitor indomethacin, the Pgp inhibitors cyclosporin A (CsA) and vinblastine, as well as conditions of energy depletion. Total calcein efflux was significantly decreased with the MRP1 inhibitors probenecid and indomethacin, while total efflux was unchanged following treatment with CsA. In the latter case, however, intracellular calcein levels postefflux were significantly greater. Probenecid and indomethacin increased calcein net secretion 2.4- and 3.5-fold, respectively. The efflux of etoposide, a known substrate for both Pgp and MRP1, was shown to be mainly Pgp-mediated by using the multidrug-resistant inhibitors quinidine (mixed Pgp/MRP1), CsA (Pgp), and MK571 (MRP1). Together, these data suggest that Calu-3 cells possess MRP1 functional activity that is subordinate to Pgp efflux. We present here kinetic analysis of calcein efflux from Calu-3 cells to support our findings.

Conjugation with L-Glutamate for in vivo brain drug delivery.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

P-glycoprotein efflux pump expression and activity in Calu-3 cells.

The purpose of this work was to determine if the sub-bronchial epithelial cell model, Calu-3, expresses the functionally active P-glycoprotein (Pgp) efflux pump. Calu-3 cells express lower levels of Pgp than both Caco-2 and A549 cells as determined by Western Blot analysis. In Calu-3 cells, accumulation of the Pgp substrates rhodamine 123 (Rh123) and calcein acetoxymethyl ester (calcein-AM) was increased in the presence of the specific Pgp inhibitors cyclosporin A (CsA), vinblastine, and taxol. Significant inhibition of Pgp activity was not observed until after 2 h in both cell lines. The organic anion/multidrug resistance associated protein-1 (MRP1) inhibitors, probenecid and indomethacin, did not affect Rh123 accumulation, whereas an increase in calcein accumulation was observed by both agents. The metabolic inhibitor sodium azide decreased the efflux of Rh123 out of Calu-3 cells to the same degree as CsA, supporting inhibition of an active, efflux pathway. The basolateral-to-apical transport of Rh123 was significantly higher than that in the reverse direction, indicating a secretory pathway of efflux that was inhibited 25-fold by CsA. Basolateral-to-apical transport of Rh123 was inhibited slightly with both MRP1 inhibitors; however, no significant effect of Rh123 net secretion was observed. Mixed inhibitor studies demonstrated that Rh123 efflux was mainly Pgp mediated. These results support an energy-dependent Pgp efflux pump pathway that is sensitive to inhibition with CsA in Calu-3 cells.

Enhancement of transport of D-melphalan analogue by conjugation with L-glutamate across bovine brain microvessel endothelial cell monolayers.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37 degrees C to 4 degrees C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

Fatty acid transport regulatory proteins in the developing rat placenta and in trophoblast cell culture models.

The placenta forms a selective barrier that is able to transport nutrients that are of critical use to the fetus. Delivery of essential fatty acids to the fetus is dependent upon transplacental transport and provides the backbone for the biosynthesis of biological membranes, myelin and various signalling molecules. The primary objective of this research was to elucidate the expression patterns of genes that regulate fatty acid transport across the placenta. Several fatty acid transport regulatory genes have been identified in the rat including; cytoplasmic heart fatty acid binding protein (hFABP), plasma membrane fatty acid binding protein (FABPpm), fatty acid translocase (FAT) and fatty acid transport protein (FATP). In this study, we have elucidated temporal and spatial expression patterns for these genes in the rat placenta and in cell culture models of the rat placenta by Northern blot, RT-PCR, Western blot and/or by in situ hybridization analyses. Expression of hFABP was specific to the labyrinth zone, the main barrier and site of transplacental transport in the rat placenta. In addition, the levels of hFABP expression increased with gestational age, suggesting a growing requirement for fatty acid transport with advancing stages of pregnancy. FABPpm, FAT and FATP are expressed in both the junctional and labyrinth zones of the rat placenta. FAT was predominantly localized to the labyrinth zone by in situ hybridization analysis. The placental cell expression patterns of the genes involved in fatty acid transport were supported by our observations of HRP-1 (labyrinth zone) and Rcho-1 (junctional zone) trophoblast cell culture models. Given their cell surface location, we predict that FABPpm, FAT and FATP potentially participate in placental fatty acid uptake. The predominant expression of hFABP and FAT in the labyrinth zone of the chorioallantoic placenta implicates hFABP and FAT in the transplacental movement of fatty acids from maternal to fetal compartments.

Carrier-mediated transport of valproic acid in BeWo cells, a human trophoblast cell line.

The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake of [14C]valproic acid by BeWo cells was found to be saturable and blocked by pre-exposure to the metabolic inhibitors, sodium azide and 2,4-dinitrophenol. Valproic acid uptake by the BeWo cells was also inhibited by the protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, but not anion exchange inhibitor. Selected monocarboxylic acids inhibited the uptake of [14C]valproic acid by BeWo cells, whereas dicarboxylic acids did not alter the uptake process. Analysis of Lineweaver-Burk plots of valproic acid uptake in the presence of benzoic acid, a marker for the monocarboxylic acid transporter, revealed a competitive process for uptake. In transcellular transport experiments, the permeation of [14C]valproic acid from the apical-to-basal side of the monolayers was significantly greater than the permeation from basal-to-apical side. Additionally, the permeation of [14C]valproic acid from apical-to-basal side was inhibited by monocarboxylic acids and not dicarboxylic acids. The results provide biochemical evidence of a proton-dependent, saturable, and asymmetric transport system, presumed to be a monocarboxylic acid transporter, for valproic acid in a human trophoblast model.

Carrier-mediated transport of monocarboxylic acids in BeWo cell monolayers as a model of the human trophoblast.

The monolayer-forming, human choriocarcinoma cell line, BeWo, was used to study the mechanisms of monocarboxylic acid transport across the human trophoblast. Benzoic acid, acetic acid, and lactic acid were used as markers for monocarboxylic acid carrier-mediated transport. The uptake of benzoic acid by BeWo cells was saturable (K(t) = 0.6 +/- 0.3 mM) at higher concentrations and significantly inhibited by typical metabolic inhibitors, sodium azide and 2, 4-dinitrophenol. A selection of different monocarboxylic acids, including a natural substrate lactic acid, also substantially inhibited the uptake of benzoic acid and acetic acid by BeWo cells, whereas dicarboxylic acids did not affect the uptake of either marker. Monocarboxylic acid uptake was pH-dependent and inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore. Kinetic analysis using Lineweaver-Burk plots revealed that monocarboxylic acids competitively inhibited the uptake of benzoic, lactic, and acetic acid by BeWo cells. In transport experiments, the permeation of benzoic acid from apical-to-basolateral side was greater than the permeation from the basolateral-to-apical side, and the transport of benzoic acid from apical-to-basolateral side was inhibited by monocarboxylic acids. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for monocarboxylic acids across the BeWo cell, a representative of the human trophoblast.

Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration.

PURPOSE: This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. METHODS: Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. RESULTS: By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. CONCLUSIONS: A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism.

Multiple cell types contribute to the pulmonary barrier including Type I and Type II alveolar epithelium. The objective of this research was to establish and characterize an in vitro model of Type II alveolar epithelium using the A549 human lung adenocarcinoma cell line. A549 cells form confluent monolayers with Type II characteristic morphology and tannic acid staining for typical lamellar bodies. A549 cells possess P450 IA1 and P450 IIB6 as determined by Western blots. Both CYPIA1 and CYPIIB6 P450 isozymes were determined to be functional with the fluorescent resorufin assay. Only the IA1 isozyme was observed to be inducible with selected polycyclic hydrocarbons. Uptake and transport experiments were carried out in cluster plates and in Snapwells. Cationized ferritin, a nonspecific absorbtive marker, was found to be taken up by the cells in a concentration-, time-, and temperature-dependent fashion. Lucifer yellow, a fluid-phase marker, was not internalized by the A549 cells. Transferrin, a representative receptor-mediated endocytic marker, was found to be taken up by the cells in a concentration-dependent and competitive fashion. Transport experiments involving fluorescein-transferrin also showed that A549 monolayers were polarized, with a greater amount of intracellular transferrin being transported out of the basolateral side of the cells. The experimental data agree favorably with literature for primary cultures of Type II pulmonary epithelial cells. These results indicated that the A549 cell line may be useful for the studying the metabolic and macromolecule processing contributions of alveolar Type II cells to mechanisms of drug delivery at the pulmonary epithelium.

Comparison of the effects of potential parenteral vehicles for poorly water soluble anticancer drugs (organic cosolvents and cyclodextrin solutions) on cultured endothelial cells (HUV-EC).

The effect of dilution of parenteral vehicles (organic cosolvent and 0.1 M cyclodextrin solutions) on cultured endothelial cells (HUV-EC) were compared in vitro. Cell morphology was observed by phase contrast light microscopy and cell viability by measuring 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction or intracellular lactate dehydrogenase (LDH) activity and total protein. Disruption of the HUV-EC monolayer was observed at dilutions of 1 in 20 for the melphalan and PEP cosolvents, 1 in 100 for an investigational drug cosolvent, and 1 in 10 for 0.1 M dimethyl-beta-cyclodextrin. In comparison, 0.1 M SBE7M- and HP-beta-cyclodextrin caused only minor disruption at a 1 in 5 dilution. MTT reduction, intracellular LDH, and total protein were decreased following exposure to 1 in 10 dilution of the melphalan cosolvent. For other test solutions, intracellular LDH activity and total protein were measured, and reductions were observed following exposure to 1 in 10, 20, and 50 dilutions of the investigational drug cosolvent and 1 in 5 dilution of DM-beta-cyclodextrin (0.1 M). At a dilution of 1 in 10, no delayed toxicity was observed for cosolvents or cyclodextrin solutions. Hence, 0.1 M SBE7M- or HP-beta-cyclodextrin formulations may be less damaging to the venous endothelium at the site of injection than organic cosolvent formulations.

Gestational and smoking effects on peptidase activity in the placenta.

Angiotensin converting enzyme (ACE) and leucine aminopeptidase (LAP) regulate fetally and maternally generated peptides in the placenta. In this study, ACE-like activity was found to be decreased and LAP-like activity increased with increasing days of gestation in rat placental tissues forming the fetal:maternal interface. Membrane-associated ACE-like and LAP-like activities in the placenta of smokers were also found to be significantly higher than their respective activities in placenta of nonsmokers. Our collective findings suggest that gestational and environmentally-induced changes in placental peptidase activities may account for variable peptide hormone and/or therapeutic peptide metabolism in the placenta.

Permeability properties of monolayers of the human trophoblast cell line BeWo.

The BeWo cell line (b30 clone) has been examined as a potential in vitro system to study transplacental transport. At the light and electron microscope level, the cells were observed to form confluent monolayers on polycarbonate filters in approximately 5 days and morphologically resembled the typical human trophoblast. BeWo monolayers developed a modest transepithelial electrical resistance and a molecular size-dependent permeability to hydrophilic passive diffusion markers, fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid permeation across BeWo monolayers was asymmetric, saturable, and inhibited by low temperature and excess competing fatty acid. Forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate treatments stimulated morphological changes in BeWo cultures and enhanced the asymmetric passage of linoleic acid across the BeWo monolayers while having minimal effects on passive permeability, affirming that the differentiation state of the cells can influence membrane transporters and transmonolayer permeability. The basic permeability properties of the BeWo monolayers suggest that the cells grown on permeable supports may be examined as a convenient in vitro system to evaluate some transplacental transport mechanisms.