Characterization of sandwich-cultured hepatocytes as an in vitro model to assess the hepatobiliary disposition of copper.

Sandwich-cultured hepatocytes (SCH) from rats (SCRH), dogs (SCDH), and humans (SCHH) were used as an in vitro model to assess the hepatobiliary disposition of copper (Cu). The expression of Cu transporters, ceruloplasmin synthesis, Cu uptake, and biliary excretion and species differences in drug-induced alterations in Cu disposition were determined in SCH from all species. Western blot analysis verified basolateral Cu uptake transporter 1 (CTR1) and canalicular Cu efflux transporter (ATP7B) expression: enzyme-linked immunosorbent assay verified synthesis/secretion of ceruloplasmin (major Cu binding protein found in blood). Endogenous Cu in SCRH, SCDH, and SCHH were 17.2 +/- 7.00, 490 +/- 44.8, and 43.5 +/- 15.8 ng/well, respectively. The hepatobiliary disposition of Cu as measured by uptake (increase in intracellular Cu in comparison to endogenous levels) and biliary excretion (increase in Cu in wash solutions obtained from hepatocytes exposed to calcium-free versus standard buffer) was determined as a function of Cu concentration and incubation time. In general, an increase in Cu concentration or incubation time resulted in an increase in Cu uptake and/or biliary excretion; however, the extent to which they affected Cu disposition was species dependent. 5-(1,1-Dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (L-000870810) (an anti-HIV compound, the development of which was halted due to an observed Cu-specific toxicity in the liver and kidneys of dogs after long-term exposure) showed no effect on Cu disposition in SCRH; however, it increased the biliary excretion of Cu in SCDH and SCHH. This is the first report to demonstrate the utility of SCH as a model to assess hepatobiliary disposition of Cu in an in vitro system.

PCB126 exposure disrupts zebrafish ventricular and branchial but not early neural crest development.

We have used zebrafish and 3,3',4,4',5-pentachlorobiphenyl (PCB126) to investigate the developmental toxicity of polychlorinated biphenyls (PCBs) that exert their effects through the aryl hydrocarbon receptor (AHR). We found that cardiac and neural crest (NC)-derived jaw and branchial cartilages are specifically targeted early in development. The suite of malformations, which ultimately leads to circulatory failure, includes a severely dysmorphic heart with a reduced bulbus arteriosus and abnormal atrioventricular and outflow valve formation. Early NC migration and patterning of the jaw and branchial cartilages was normal. However, the jaw and branchial cartilages failed to grow to normal size. In the heart, the ventricular myocardium showed a reduction in cell number and size. The heart and jaw/branchial phenotype could be rescued by pifithrin-alpha, a blocker of p53. However, the function of pifithrin-alpha in this model may act as a competitive inhibitor of PCB at the AHR and is likely independent of p53. Morpholinos against p53 did not rescue the phenotype, nor were zebrafish with a mutant p53-null allele resistant to PCB126 toxicity. Morpholino knockdown of cardiac troponin T, which blocks the onset of cardiac function, prevented the PCB126-induced cardiac dysmorphogenesis but not the jaw/branchial phenotype. The cardiovascular characteristics appear to be similar to hypoplastic left heart syndrome (HLHS) and introduce the potential of zebrafish as a model to study this environmentally induced cardiovascular malformation. HLHS is a severe congenital cardiovascular malformation that has previously been linked to industrial releases of dioxins and PCBs.

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus).

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.

The Immunoglobulin G Heavy Chain (IGHG) genes of the Atlantic bottlenose dolphin, Tursiops truncatus.

Dolphin Immunoglobulin G Heavy Chain (IGHG) sequences were obtained by PCR amplification of cDNA from peripheral blood leukocytes using degenerate primers. Analysis of full-length sequences indicated the presence of two expressed isotypes, IGHG1 and IGHG2 that differ mainly in the hinge region of the molecule. Genomic Southern blot analysis indicated that the IGHG1 and IGHG2 genes are most likely present in single copies. The inferred amino acid sequences show greatest similarity between the dolphin and other closely related artiodactyl species. The genetic structure of the IGHG genes were deduced through genomic PCR and revealed that the hinge regions of both IGHG1 and IGHG2 are encoded by a single exon. The transmembrane region of the dolphin IGHG chain shows similarity to the transmembrane region of other mammalian IGHG chains with a canonical CART motif. This is in contrast to the unusual Ser to Gly substitution previously found in the dolphin IGHM transmembrane region, and the functional significance of this variation for B cell antigen-receptor dimer activation remains unknown.

Pharmacokinetics of olanzapine after single-dose oral administration of standard tablet versus normal and sublingual administration of an orally disintegrating tablet in normal volunteers.

Olanzapine (OLZ) is a second-generation antipsychotic agent available in 2 solid oral dosage forms, a standard oral tablet (SOT) and an orally disintegrating tablet (ODT). This study assessed the absorption of each by different routes of administration. Secondarily, the influence of P-glycoprotein (P-gp) genotype was assessed. It was hypothesized that more rapid absorption of the OLZ ODT would occur when administered sublingually versus standard oral administration. A randomized, 3-way crossover study assessed the 5-mg OLZ formulations in healthy volunteers (n = 10). Blood was collected (0-8 hours) to assess OLZ pharmacokinetics using liquid chromatography/mass spectrometry. Both routes of ODT administration resulted in more measurable early concentrations relative to SOT. However, there were no statistically significant differences observed between any of the OLZ exposures for observed pharmacokinetic parameters (C(max), T(max), AUC(0-8h)). The homozygous TT genotype for P-gp resulted in an increased AUC of OLZ for SOT administration but not for either condition where sublingual absorption could occur.

Characterization of P-glycoprotein inhibition by major cannabinoids from marijuana.

The ATP-dependent drug efflux transporter P-glycoprotein (P-gp) plays a significant role in the absorption and disposition of many compounds. The purpose of this study was to investigate the possible interaction of P-gp with each of four major marijuana constituents: Delta(9)-tetrahydrocannabinol (THC), 11-nor-Delta(9)-tetrahydrocannabinol-carboxylic acid (THC-COOH), cannabinol (CBN), and cannabidiol (CBD). The results of a P-gp ATPase activity screen showed that THC-COOH, CBN, THC, and CBD all stimulated P-gp ATPase activity with a Michaelis-Menten parameter (V(max)/K(m)) value of 1.3, 0.7, 0.1, and 0.05, respectively. Furthermore, CBD showed a concentration-dependent inhibitory effect on verapamil-stimulated ATPase activity with an IC(50) value of 39.6 microM, whereas all other tested cannabinoids did not display appreciable inhibitory effects. Thus, the inhibitory effects of CBD on P-gp transport were further studied. At concentrations ranging from 5 to 100 microM, CBD robustly enhanced the intracellular accumulation of known P-gp substrates rhodamine 123 and doxorubicin in a concentration-dependent manner in Caco-2 and LLC-PK1/MDR1 cells. An IC(50) value of 8.44 microM was obtained for inhibition of P-gp function in LLC-PK1/MDR1 cells as determined by flow cytometry using rhodamine 123 as a fluorescence probe. Following exposure to 30 microM CBD, the apparent permeability coefficient of rhodamine 123 across Caco-2 and rat brain microvessel endothelial cell monolayers was increased to 2.2- and 2.6-fold in the apical-to-basolateral direction but decreased to 0.69- and 0.47-fold in the basolateral-to-apical direction, respectively. These findings indicate that CBD significantly inhibits P-gp-mediated drug transport, suggesting CBD could potentially influence the absorption and disposition of other coadministered compounds that are P-gp substrates.

Multiple night-time doses of valerian (Valeriana officinalis) had minimal effects on CYP3A4 activity and no effect on CYP2D6 activity in healthy volunteers.

Valerian (Valeriana officinalis) is a popular dietary supplement. The objective of this study was to assess the influence of a valerian extract on the activity of the drug-metabolizing enzymes cytochrome P450 2D6 (CYP2D6) and 3A4. Probe drugs dextromethorphan (30 mg; CYP2D6 activity) and alprazolam (2 mg; CYP3A4 activity) were administered orally to healthy volunteers (n = 12) at baseline and again after exposure to two 500-mg valerian tablets (1000 mg) nightly for 14 days. The valerian supplement contained a total valerenic acid content of 5.51 mg/tablet. Dextromethorphan to dextorphan metabolic ratios (DMRs) and alprazolam pharmacokinetics were determined at baseline and after valerian treatment. The DMR was 0.214 +/- 0.025 at baseline and 0.254 +/- 0.026 after valerian supplementation (p > 0.05). For alprazolam, the maximum concentration in plasma was significantly increased after treatment with valerian (25 +/- 7 ng/ml versus 31 +/- 8 ng/ml; p < 0.05). There were no significant differences in other pharmacokinetic parameters at baseline and after valerian exposure (all p values > or = 0.05; time to reach maximum concentration in plasma, 3.0 +/- 3.2 versus 3.1 +/- 2.1 h; area under the plasma concentration versus time curve, 471 +/- 183 versus 539 +/- 240 hx ng x ml(-1); half-life of elimination, 13.5 +/- 4.3 versus 12.2 +/- 5.6 h). Our results indicate that although a modest increase was observed in the alprazolam Cmax, typical doses of valerian are unlikely to produce clinically significant effects on the disposition of medications dependent on the CYP2D6 or CYP3A4 pathways of metabolism.

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage.

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.